Peptides that target dorsal root ganglion neurons

ABSTRACT

The present invention concerns methods and compositions that employ peptides that target dorsal root ganglion (DRG) neurons. In particular, the peptides are used to target therapeutic agents, such as proteins, liposomes, or viral particles comprising therapeutic polynucleotides, to one or more peripheral neuropathies or neuropathic pain, for example. In particular cases, the peripheral neuropathies or neuropathic pain is caused directly or indirectly by DRG neuronopathy.

This application claims priority to U.S. patent application Ser. No.12/864,895, filed Jul. 28, 2010, which was a US §371 application of PCTPatent Application Serial No. PCT/US2009/032643, filed Jan. 30, 2009,which claims priority to U.S. Provisional Patent Application Ser. No.61/024,892, filed Jan. 30, 2008, which is all of which applications areincorporated by reference herein in its their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under NationalInstitutes of Health Grant No. HL-51586 and HL-59314. The United StatesGovernment has certain rights in the invention.

TECHNICAL FIELD

The present invention generally concerns at least the fields ofneurology, cell biology, molecular biology, and medicine. In particular,the present invention concerns the field of dorsal root ganglion biologyand treatment of diseases related thereto.

BACKGROUND OF THE INVENTION

Phage display is a powerful technology to identify peptide sequencemotifs that target a particular tissue or cell type in the body (Arap etal., 2002; Kolonin et al., 2004; Sidhu, 2001). Coupling such peptides todrugs and genes would enable their targeted delivery to specific cellsand tissues in vitro and in vivo (Arap et al., 2002; Petty et al., 2007;Sergeeva et al., 2006; White et al., 2004). A commonly used platform isthe combinatorial filamentous M13 phage library that displays shortrandom peptides fused to a minor coat protein (pIII) (Sidhu, 2001),which can be used to isolate specific cell type-binding peptides by aprocedure called biopanning (Petty et al., 2007). In the presentinvention, the technology has been applied to identify peptide motifsthat recognize, and are specifically taken up by, neurons in the dorsalroot ganglion (DRG) in mice as an exemplary model for humans. DRGneurons are target cells for the treatment of diseases of the peripheralsensory nervous system (Sghirlanzoni et al., 2005), in certainembodiments of the invention For instance, neuropathic pain is a commonsymptom in various disorders, including metabolic abnormalities,malignancies, physical injuries, toxins and poisons, and hereditarydiseases (Sghirlanzoni et al., 2005), and is the cause of much morbidityand misery. Although various pharmaceutical agents, anesthetics,surgical operations, or procedures such as transcutaneous electricalnerve stimulation, have been used to treat the symptoms of neuropathicpain (Mendell and Sahenk, 2003), such palliative treatments are mostlynon-targeted and of limited efficacy (Mendell and Sahenk, 2003). DRGneurons are the primary afferent neurons and can be classified into twobroad groups: large and small neurons. Large neurons are thought to beinvolved mainly in proprioception, while most small neurons are involvedin nociception (Zhang and Bao, 2006). Both neuronal populations havealso been subclassified by biochemical and histological methods, such aslineage tracing or immunostaining of different markers(neurotransmitters, cell surface carbohydrates) (Dodd and Jessell, 1985;Kusunoki et al., 1991; Marmigere and Ernfors, 2007; Price and Flores,2007). The present invention provides novel therapeutics and treatmentmethods for disease processes affecting the peripheral sensory nervoussystem (Goss, 2007).

Dysfunction of neurons in dorsal root ganglion (DRG) occurs in a varietyof sensory neuronopathies (Sghirlanzoni et al., 2005; Kuntzer et al.,2004), including hereditary (Swanson et al., 1965), autoimmune (Malinowet al., 1986), nutritional (Montpetit et al., 1988), metabolic (Yasudaet al., 2003) and neoplastic diseases (Wanschitz et al., 1997). DRGneuronal disorders are associated with neuropathic pain, loss ofsensation and sensory ataxia (Sghirlanzoni et al., 2005; Kuntzer et al.,2004). Delivery of neurotrophic factors can minimize neuronal damage inthe DRG (Chattopadhyay et al., 2005; Wang et al., 2005; Pezet et al.,2006). However, neurotrophic polypeptides are susceptible to proteolyticdegeneration and their therapeutic effects are short-lived; directdelivery of therapeutic genes proves to be more effective (Xu et al.,2003). Herpes simplex virus (Chattopadhyay et al., 2005) and poliovirus(Jackson et al., 2003) injected through subcutaneous or intramuscularroutes are taken up by endocytosis at nerve terminals and travel viaaxonal transport to somas of DRG neurons. While the approach works innormal functioning nerves, the efficacy of such a strategy is greatlycompromised under disease conditions. To circumvent this problem, directinjection into DRG neurons by microneurosurgery can be accomplished bythe removal of a piece of vertebra to gain access to the DRG (Xu et al.,2003; Glatzel et al., 2000). Such a maneuver is, however, not practicalfor repeated injections, which is likely required for chronic disorders.In contrast, intrathecal (IT) injection is a far less invasive procedurethat is used routinely in clinical practice (Wang et al., 2005).Nevertheless, DRGs are ensheathed in the dura and bathed incerebrospinal fluid (CSF). Gene delivery vectors could disperse and betaken up elsewhere leading to complications, including meningitis(Driesse et al., 2000). To optimize the chance for successful DRG neurondelivery while minimizing undesired off-target effects, targeted genedelivery would be an ideal strategy for delivering genes to DRG neurons(Waehler et al., 2007).

Adenovirus (Ad) is an efficient gene transfer system in vitro as well asin vivo because of its capacity to infect both quiescent andproliferating cells, a broad spectrum of tissue transductionsusceptibility and relative ease with which the vector can be producedin high titer. Helper-dependent adenoviral vector (HDAd) is the mostadvanced Ad, which is devoid of all viral coding sequences. The lack ofpotentially harmful Ad viral gene expression is associated with markedlyreduced toxicity. HDAd-mediated in vivo gene delivery in rodents andnonhuman primates has been found to have an excellent safety profile andprotracted transgene expression (Seiler et al., 2007; Brunetti-Pierriand Ng, 2008). Other advantages of HDAd are a large cloning capacity (upto 37 kb) and a greatly attenuated adaptive host immune response (Muruveet al., 2004). Thus, in specific embodiments the invention concerns HDAdas the backbone for the targeted vector for DRG neuron delivery.

The cellular tropism of Ad serotype 5 (Ad5) is determined by thecell-surface expression of primary attachment sites, the Coxsackie andAdenovirus receptor (CAR) (Bergelson et al., 1997; Tomko et al., 1997)and heparan sulfate proteoglycans (HSPG) (Dechecchi et al., 2001) and/orlow-density lipoprotein receptor-related protein, and via the integrins,which act as secondary internalization receptors (Shayakhmetov et al.,2005; Koizumi et al., 2006; Parker et al., 2006). Cells deficient in theexpression of these receptors are transduced at a greatly reducedefficiency. Little or no CAR is expressed by DRG neurons, which are poortargets for unmodified Ads (Hotta et al., 2003). Considerable effort hasbeen expended on the generation of first generation Ads that targetnormally refractory tissues, as well as their retargeting to alternativecell-type specific receptors. Three major strategies have been employedin this respect: genetic modification of Ad capsid proteins, conjugationof adaptor proteins such as antibody, or bispecific fusion proteins, andchemical modification by polymers with targeting ligands (Waehler etal., 2007; Mizuguchi and Hayakawa, 2004). Genetic modification appearsto the most popular of these approaches. This technology is particularlyappealing for HDAd because capsid proteins of HDAd are supplied by thehelper virus (HV). Armed with a library of targeting HVs, one canproduce HDAds that potentially target any tissue by applying thespecific HV at the final amplification. To date, despite the manypotential advantages of HDAd, targeting HDAd by genetic modification hasbeen limited to the introduction of peptide ligands into the HI loop(Biermann et al., 2001) or replacement of fiber gene with that of adifferent Ad serotype (Wang et al., 2005).

BRIEF SUMMARY OF THE INVENTION

In general, the present invention concerns methods and compositions forthe treatment of medical conditions that involve dorsal root ganglionneurons, including specific subtypes thereof. In specific aspects of theinvention, the present invention concerns methods and compositions fortreating peripheral neuropathies, as well as neuropathic pain, forexample. In particular embodiments, the present invention concerns anovel strategy for the treatment of sensory neuronopathies, for exampleusing a sensory neuron-targeted helper-dependent adenoviral vector

In certain embodiments, there are peptides that home to mouse dorsalroot ganglion (DRG). These peptides were obtained from an exemplaryphage library expressing random 7-mer peptides fused to a minor coatprotein (pIII) of the M13 phage. An in vitro biopanning procedureyielded 113 phage plaques after five cycles of enrichment by incubationwith isolated DRG neurons and two cycles of subtraction by exposure toirrelevant cell lines. Analyses of the sequences of this collectionidentified three peptide clones that occurred repeatedly during thebiopanning procedure.

Phage-antibody staining revealed that the three exemplary peptides boundto DRG neurons of different sizes. The peptides were injected asindividual GST-peptide fusion proteins into the subarachnoid space ofmice and there was the appearance of immunoreactive GST in the cytosolof DRG neurons with a similar size distribution as that observed invitro, indicating that the GST-peptide fusion proteins were recognizedand taken up by different DRG neurons in vivo. The identification ofhoming peptide sequences provides a powerful tool for future studies onDRG neuronal function in vitro and in vivo, and opens up the possibilityof neuron-specific drug and gene delivery in the treatment of diseasesaffecting DRG neurons.

In additional embodiments, an enzyme gene was delivered to mice with theenzyme deficiency (hexB-ko). These mice have peripheral neuropathy andabnormal functional tests, although they are known in the art for havingmental retardation but not peripheral neuropathy (the inventors testedif they had neuropathy because it can objectively be measured). Thetreatment significantly ameliorated the functional deficiencies. Thepeptide was engineered to the coat of HDAd, and there was homing to DRGneurons. Therefore, the inventors used the re-targeted HDAd to deliverthe hexB to the subarachnoid space and successfully targeted the DRGneurons. Wildtype Ad delivery did not work.

In one embodiment of the invention, there is a method of targeting adorsal root ganglion neuron in an individual, comprising the step ofdelivering to the individual one or more peptides from Table 2, or aderivative thereof. In a specific embodiment, the peptide is selectedfrom the group consisting of SPGARAF (SEQ ID NO:1), DGPWRKM (SEQ IDNO:2), FGQKASS (SEQ ID NO:3), or a mixture thereof. In an additionalembodiment, the individual has a peripheral neuropathy or neuropathicpain.

In certain embodiments of the present invention, methods andcompositions are employed to engineer fiber-modified HDAd anddemonstrate the efficacy of HDAd bearing DRG neuron-specific targetingpeptide (Oi et al., 2008) to correct a genetic deficiency and reverseits associated sensory deficit in a mouse model (Sango et al., 1995;Sango et al., 2002). Targeting ligands were introduced into the fiber ofHV lacking the CAR and HSPG binding sites and showed that HV lackingprimary attachment sites has markedly impaired ability to transduce 293cells and DRG neurons in vitro, and that addition of DRG targetingpeptide ligands efficiently retargets HV to DRG neurons. In addition,using Cre-expressing HDAd amplified in the presence of fiber-modifiedHVs, there was high efficiency DRG neuron targeting in vivo followingintrathecal injection into ROSA-GFP transgenic mice. Finally, efficacyof the targeting strategy to correct a genetic deficiency in DRG neuronswas demonstrated, reversing specifically the peripheral sensory nervoussystem dysfunction in mice with β-hexosaminidase deficiency. In certainembodiments of the invention, targeted HDAd gene therapy for DRG neurondisorders is useful.

In one embodiment of the invention, there is an isolated peptide of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, or a modified peptide having one,two, or three conservative substitutions compared to SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3, respectively, or a combination thereof. In aspecific embodiment, the peptide is comprised in a pharmaceuticallyacceptable excipient. In a particular embodiment, the peptide is linkedto a protein, such as a therapeutic protein, or a liposome. In certainaspects, the protein is present on the surface of a helper-dependentadenoviral particle. In particular cases, the viral particle is definedas further comprising a therapeutic polynucleotide.

In another embodiment of the invention, there is a method of targeting adorsal root ganglion (DRG) neuron in an individual, comprising the stepof delivering to the individual an effective amount of one or morepeptides of the invention. In a specific embodiment, the individual hasDRG neuronopathy. In certain cases, the DRG neuronopathy results in aneuropathy. In particular cases, the individual has pain, hyperalgesia,hypoalgesia, or ataxia. In specific embodiments, the helper-dependentadenoviral particle is delivered intrathecally.

In a certain embodiment of the invention, there is a method of treatinga DRG neuronopathy in an individual, comprising the step of deliveringto the individual an effective amount of a therapeutic agent linked toone or more peptides of the invention or an effective amount of aliposome comprising a therapeutic agent, wherein the is liposome linkedto one or more peptides of the invention.

In an additional embodiment, there is a method of treating a DRGneuronopathy in an individual, comprising the step of delivering to theindividual an effective amount of one or more peptides of the invention.In certain cases, the DRG neuronopathy results in a neuropathy. Inparticular cases, the individual has pain, hyperalgesia, hypoalgesia, orataxia. In specific embodiments, the helper-dependent adenoviralparticle is delivered intrathecally. In certain cases, the invention isutilized for sensory neuropathy and/or sensory neuronopathy.

In certain embodiments, there is one or more polynucleotides thatencodes a peptide or a peptide and protein of the invention. In certainaspects, the peptide is linked covalently to the protein, and inparticular cases the peptide and protein are fused as a proteinaceousmolecule and/or are encoded by a single polynucleotide.

In particular embodiments of the invention, a therapeutic protein isNGF, EGF, FGF, BDNF, IGF1, CTNF, PDNF, VAD, DEVD, NGN1, NGN2, NGN3,RUNX3, or a signal peptide. In specific embodiments, a therapeuticpolynucleotide that encodes a therapeutic protein is selected from thegroup consisting of NGF, EGF, FGF, BDNF, IGF1, CTNF, PDNF, VAD, DEVD,NGN1, NGN2, NGN3, RUNX3, IL-10, anti-TNFαc, EPO, or prepro-β endorphin.

In specific aspects of the invention, DRG neuronopathy results in aneuropathy selected from the group consisting of neuropathies associatedwith metabolic disease, hereditary disease, neoplasm, infectiousdisease, injury, or in autoimmune disease. In a specific embodiment, theindividual has pain, hyperalgesia, hypoalgesia, or ataxia. In aparticular embodiment, the helper-dependent adenoviral particle isdelivered intrathecally.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated that the conception and specific embodimentdisclosed may be readily utilized as a basis for modifying or designingother structures for carrying out the same purposes of the presentinvention. It should also be realized that such equivalent constructionsdo not depart from the invention as set forth in the appended claims.The novel features which are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1 shows binding of 3 different phages to cultured-DRG neurons. Thecells were analyzed by a confocal laser-scanning microscopy. (A) Cellsize distribution of the DRG neurons positive for DRG-p1, DRG-p2 orDRG-p3. (B) Bindings of the 3 different phage clones to mouse DRGneurons. Positive reactions stained with anti-fd antibody (Ab) aregreen. Ab (−) is without antibody and Ab+peptide is antibody pluspeptides (10⁻⁴ M) corresponding to each phage. Nuclei were stained withPI (red). Bars=20 μm.

FIG. 2 demonstrates fluorescence microscopy of in vivo binding ofGST-peptide-fusion proteins to DRG neurons. (A) Cell size distributionof the DRG neurons positive for GST-DRG1, GST-DRG2 or GST-DRG3. (B)Double immunofluorescence staining of DRG-neurons with anti-GST (green)and anti-neurofilament L (NF-L, non-phosphorylated form, red). Nucleiwere stained with DAPI (blue). None indicates no primary antibody.Arrows indicated the cells positive for both GST and NF-L. Bars=20 μm.

FIG. 3 Strategy for construction of fiber-modified helper-dependentadenoviral vectors (HDAd). (a) Construction of fiber-modified helpervirus (HV). HV is a first generation El-deleted Ad vector containing thepackaging signal flanked by loxP sequences. This HV provides all of thecomponents necessary for replication and packaging of HDAd genome intrans, but cannot be packaged by itself upon coinfection of Creexpressing packaging cells with HDAd due to the excision of thepackaging signal for HV. The first step to construct targeted HDAd is toproduce fiber-modified HV. In the approach, HV is detargeted by ablationof primary docking sites, CAR and HSPG, and then adding a targetingpeptide ligand. HV produced by transfection contains no primary dockingsites to 293 cells for infection and is unable to reinfect foramplification. To overcome this problem, 293 cells were establishedexpressing Ad serotype 5 wild type fiber (293-fiber). HV produced on293-fiber has both wild type and modified fiber and can infect 293 cellseffectively while the modified fiber genome is maintained. But, the wildtype fiber can be removed by infection of 293 cells. (b) Construction ofHDAd. The genome of HDAd contains only essential cis-acting elements(inverted terminal repeats for replication and the packaging signal) andis introduced into packaging cells expressing Cre by transfection. Bycoinfection with HV containing wild type fiber, HDAd genome is packagedinto Ad particles. HV continues to provide all of the componentsnecessary for replication and packaging, but the HV genome cannot bepackaged. (c) Generation of fiber-modified HDAd. Final step ofproduction of fiber-modified HDAd is coinfection of 293Cre with HDAd andHV made on 293-fiber cells. HDAd genome is packaged into viral particlesencoded by HV genome, which results in HDAd capsids containing modifiedfiber.

FIG. 4 Transduction of DRG neurons by fiber-modified helper virus (HV).(a) Features of modified fiber proteins. WF contained Ad5 wild typefiber. All other HVs had the KO1S* backbone which has the BspEI site(blue letters) within 6 amino acids linker (fourth row; SEQ ID NO:6) andmodification of heparan sulfate proteoglycan (HSPG) binding site (KKTK;corresponding sequence is SEQ ID NO:104) to GAGA (second row;corresponding sequence is SEQ ID NO:105) and Coxsackie and Adenovirusreceptor (CAR) binding site (SP) to EA (third row). DRG homing peptides(DRG1, DRG2 and DRG3) were inserted into the BspEI site in the HI loopof fiber protein (bottom), with insert sequences corresponding to SEQ IDNO:107, SEQ ID NO:108, and SEQ ID NO:109, respectively. Numbers show theposition of amino acid in the fiber protein. (b) Luciferase activity in293 cells. 293 cells were infected with HVs at 1,000 viral particles(vp)/cell and were harvested 24 hours after infection for luciferaseassay. Results are expressed as relative light units (RLU) per mgprotein (n=6). (c) Luciferase activity in HIV infected DRG neurons.Primary DRG neurons were infected and luciferase activity was determinedas described in (b). (d) LacZ expression in nervous system. 1×10⁸ vp offiber modified HVs were injected into C57BL/6 mice through subarachnoidspace at lumbar level. Mice were sacrificed 5 days after HV injectionand stained for LacZ expression. Upper panels show whole DRG tissueswith nerves and lower panels showed brain and spinal cord. Scale bar, 2mm. Arrows in the WF group indicate X-gal positive areas at nerveportion in upper panel and at dorsal intermediate sulcus of spinal cordin lower panel. Arrowhead shows the position of vector injection. (e)Luciferase activities in nervous systems. 1×10⁸ vp of fiber-modified HVswere injected into C57BL/6 mice as described in (d) and neuronal tissueswere harvested 5 days after injection. (f) Long term LacZ expression inDRG. Mice were treated with 1×10⁸ vp of HV-WF or -DRG1 and weresacrificed at various time. LacZ mRNA in DRG was quantified by real timeRT-PCR and normalized to b-actin mRNA. Data are expressed as relative tothe LacZ mRNA in the HV-WF group at day 60. *P<0.001, **P<0.01 and***P<0.05 vs. WF group.

FIG. 5 Generation of 293-fiber cell and structure of fiber-modifiedhelper viruses (HVs). (a) Immunoblot analysis of 293 cells expressingadenovirus serotype 5 (Ad5) fiber protein. (upper panel). Upper panel:The 62 kDa band corresponds to the monomer of fiber protein. Highmolecular size bands are the predicted trimer of the fiber protein. Theclone 15 highlighted in red was further characterized forcomplementation of wild type fiber for HV amplification. Lower panel:structure of lentiviral vector expressing Ad5 wild type fiber. (b)Nucleotide sequence of the HI loop in fiber-modified HV genome. DNA wasextracted from purified HV and characterized by DNA sequence analysis.*Nucleotide change from T to G as a result of subcloning targeting motifinto the BspEI site. Illustrated sequence for HV-KO1S* is SEQ ID NO:111;for HV-DRG1 is SEQ ID NO:112; for HV-DRG2 is SEQ ID NO:113; and forHV-DRG3 is SEQ ID NO:114.

FIG. 5 Generation of 293-fiber cell and structure of fiber-modifiedhelper viruses (HVs). (a) Immunoblot analysis of 293 cells expressingadenovirus serotype 5 (Ad5) fiber protein. (upper panel). Upper panel:The 62 kDa band corresponds to the monomer of fiber protein. Highmolecular size bands are the predicted trimer of the fiber protein. Theclone 15 highlighted in red was further characterized forcomplementation of wild type fiber for HV amplification. Lower panel:structure of lentiviral vector expressing Ad5 wild type fiber. (b)Nucleotide sequence of the HI loop in fiber-modified HV genome. DNA wasextracted from purified HV and characterized by DNA sequence analysis.*Nucleotide change from T to G as a result of subcloning targeting motifinto the BspEI site.

FIG. 6 LacZ expression in cells following infection with fiber-modifiedhelper virus in vitro. (a) X-gal stain of 293 cells 24 hours afterinfection with fiber-modified HVs at 1−1×10⁴ vp/cell. Bar, 50 mm. (b)X-gal stain of DRG neurons. Bar, 20 mm.

FIG. 7 X-gal stain of nervous tissues. 1×10⁸ vp of fiber-modified HVswere injected into wild type C57/BL6 mice via subarachnoid space at thelumbar level. Mice were sacrificed for X-gal staining 5 days after HVinjection. (a) Brain. Arrow shows X-gal stain in the lateral ventricularregion. Bar, 1200 mm. (b) Spinal cord. Arrow shows central canal ofspinal cord. Bar, 250 mm. (c) DRG. Bar, 100 mm. (d) Sciatic nerve.Arrows indicate X-gal positive areas at the surface of nerves. Bar, 50mm.

FIG. 8 Helper-dependent adenoviral vectors harboring DRG neuron homingpeptides transduce DRG neurons and turn on GFP expression in ROSA-GFPtransgenic mice. (a) A schematic presentation of the strategy toevaluate targeting tissues in ROSA-GFP transgenic mice usingfiber-modified HDAd expressing Cre. In ROSA-GFP mice, GFP geneexpression is blocked by a STOP fragment flanked by loxP sequence. Uponexcision of the STOP fragment by Cre expression, GFP is expressed.Thereby, GFP expression is dependent on HDAd-mediated Cre gene transfer.(b,c) Cre and GFP mRNA expression in neuronal tissues. 1×10⁸ vp offiber-modified HDAd expressing Cre were injected through subarachnoidspace at lumbar level into ROSA-GFP mice. Neuronal tissues wereharvested 5 days after vector injection and mRNA was quantified by realtime RT-PCR. Results are expressed as relative to the mRNA level in DRGof mice treated with HDAd containing wild type fiber. *P<0.05 and**P<0.01 vs. WF group, #P<0.05, ##P<0.01 vs. KO1S* group. HVcontamination in fiber-modified HDAd determined by real time PCR rangedfrom 0.0007 to 0.037%. (d) Immunoblot analysis of Cre and GFP proteinexpression 5 days after HDAd injection. 20 μg protein was loaded on7.5-15% SDS-PAGE. Band intensities were normalized to GAPDH. Proteinexpression is expressed relative to that of HDAd-WF. (e) GFP expressionin DRG. Upper and middle panels show whole DRG tissues. Upper panel isunder GFP filter and the middle is under bright filter. Broken linesoutline the margin of DRG tissues. Bottom panel is DRG sections withDAPI nuclear stain. Bar, 100 μm. (f,g) Long term Cre and GFP mRNAexpression in DRG. 1×10⁸ vp of HDAd-WF-Cre or HDAd-DRG1-Cre wereinjected into ROSA-GFP mice and sacrificed at various time points forquantitation of Cre and GFP mRNAs in DRG. The mRNA levels are expressedas relative to the mRNA in HDAd-WF-Cre-treated mice sacrificed at day60. *P<0.001, **P<0.01 vs. WF group.

FIG. 9 Histological analysis of GFP expression in brain, spinal cord andsciatic nerve isolated from ROSA-GFP mice after administration offiber-modified HDAd. (a-c) Whole tissue image. First row shows the imageunder GFP filter. Second row shows the same area under bright field.Bar, 2 mm in a, 500 mm in b, 200 mm in c.

FIG. 10 Fiber-modified HDAd supports long-term transgene expression andimproves safety profile. (a) Long term expression of lacZ gene in DRG.1×108 of Ad vectors were injected into C57BL/6 wild type mice and DRGwas isolated at various times for quantitation of LacZ mRNA. The LacZmRNA levels were normalized to β-actin mRNA. All results are expressedas relative to LacZ mRNA level at day 5. *P<0.001, **P<0.01 vs. HV withthe same fiber (e.g., HDAd-WF vs. HV-WF). (b-d) Targeted HDAd vectorinduces less inflammation. Cerebrospinal fluid (CSF) was collected atvarious time points and cytokines were measured by ELISA. *P<0.01 and**P<0.05 vs. HV-WF, ^(†)P<0.01 and ^(\\)P<0.05 vs. HDAd-WF, \P<0.01 and\\P<0.05 vs. HV with the same fiber.

FIG. 11 Cytokine levels in cerebrospinal fluid (CSF). PBS was injectedto C57BL/6 mice through subarachnoid space at lumbar level. CSF wascollected at 3 hour, 24 hour and day3. Cytokines were measured by ELISA.a. IL-6, b. TNF-a and c. IL-1b. *P<0.01 and **P<0.05 vs. no injection(n=3−4).

FIG. 12 Gene therapy for a mouse model of Sandhoff disease. (a) hexbgene expression in neuronal tissues. 1×10⁸ vp of various fiber-modifiedHDAd vectors were injected into hexb−/− mice through subarachnoid spaceat lumbar level and neuronal tissues were isolated for extraction ofcellular RNA 8 weeks after injection. The hexb mRNA was quantified byreal time RT-PCR and relative hexb mRNA expression to that in brain ofhexb+/+ mice was calculated. *P<0.001, **P<0.05. (b-d) Hexosaminidase(Hex) activities (total in b, Hexosaminidase A (HexA) in c andHexosaminidase B (HexB) in d) in nervous tissues from hexb+/+ andhexb−/− mice with injection. HexB activity was calculated by subtractinghexA activity from total hex activity. *P<0.001, **P<0.01. \P<0.01 and\\P<0.05 vs. hexb−/− treated with HDAd-empty. (e-h) Immunofluorescencefor Hexb protein in DRG. (i-1) In situ staining for hexosaminidaseactivity. Nuclear was stained with methyl green. Arrows indicate weakpositive staining in neurons. Bar, 20 μm.

FIG. 13 DRG-targeted hexb expression rescues sensory neuronal responsesin hexb−/− mice. Electrophysiological studies and behavior tests wereperformed at 4 and 8 weeks after treatment. (a) Sensory nerve conductionvelocity was measured on sural nerve. (b) Sensory nerve action potentialwas recorded in proximal site after stimulation in the distal site atankle joint. (c) First contact time. (d) Adhesive removal test. Data areexpressed as means±S.D. (n=8−12). *P<0.01, **P<0.05. \P<0.01 and\\P<0.05 vs. hexb+/+ mice. (e-h) Niss1 stain. (i-1) PAS stain withhematoxylin for nuclear stain. (m-p) Enlargement of black square in i-1.Arrows indicate granular formations in neurons. Bar, 20 μm.

FIG. 14 Motor nerve conduction velocity (MNCV) and compound muscleaction potential (CMAP) in hexb−/− mice after treatment withfiber-modified HDAd expressing hexb. (a,b) Measurement was performed at4 and 8 weeks after injection. #P<0.05 vs. hexb+/+ mice (n=8−12).

FIG. 15 PCR analysis for excision of the packaging signal from helpervirus. (a) Excision of the packaging signal upon infection of 293Crecells. Left panel: PCR of purified HV genome. PCR was performed usingthe following primers corresponding to 5′ and 3′ flanking sequence tothe packaging signal: 5′-primer, 3′-primer. The expected PCR product is460 bp. Right panel: HV genome extracted from CVL after infection of293-cre cells. Due to excision of the packaging signal, the size of PCRproduct is reduced to 262 bp. (b) Nucleotide sequence of HV genomebefore (upper panel) and after (lower panel) infection of 293-cre cells.See Erturk et al. (2003).

DETAILED DESCRIPTION OF THE INVENTION

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. Still further,the terms “having”, “including”, “containing” and “comprising” areinterchangeable and one of skill in the art is cognizant that theseterms are open ended terms. Some embodiments of the invention mayconsist of or consist essentially of one or more elements, method steps,and/or methods of the invention. It is contemplated that any method orcomposition described herein can be implemented with respect to anyother method or composition described herein.

I. General Embodiments of the Present Invention

In the present invention, the inventors have isolated three exemplarypeptides that recognize specific, defined sizes of DRG neurons. Thesepeptides are useful for targeting DRG neurons and for therapeutics fordiseases involving specific subtypes of DRG neurons, for example.

In certain aspects, the invention concerns methods and compositions thattarget therapeutic genes to dorsal root ganglion (DRG) neurons for thetreatment of DRG neuronopathy, a common disorder for which there is nosatisfactory treatment. The inventors inserted DRG homing-peptides tothe fiber of helper virus (HV), an adenovirus (Ad) that is detargetedfor binding to Coxsackie and Ad receptor and heparan sulfateproteoglycans, and used it to generate DRG-targeting helper-dependent Ad(HDAd). Intrathecal injection of the HDAd produced a 100-fold highertransduction of DRG neurons and a markedly attenuated inflammatoryresponse as compared to unmodified HDAd. The inventors tested thistreatment strategy in a disease model by injecting HDAd-β-hexosaminidaseβ-subunit to β-hexosaminidase-deficient mice. Delivery of the targetingHDAd reinstated neuron-specific β-hexosaminidase production, reversedthe gangliosidosis, and ameliorated peripheral sensory dysfunction. Thedevelopment of DRG neuron-targeted HDAd with proven efficacy is usefulfor the treatment of sensory neuronopathies of diverse etiologies.

II. Peptides of the Invention

In certain embodiments, the present invention concerns novelcompositions comprising at least one peptide. In one embodiment of theinvention, there is an isolated peptide from Table 2, for example of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, or a modified peptide thereof havingone, two, three, four, or more conservative substitutions compared toSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, respectively; or a combinationthereof.

As used herein, a peptide is a composition of from about 3 to about 25amino acids, although in specific embodiments the peptide is between 5and 20 amino acids, 5 and 15 amino acids, 5 and 10 amino acids, 6 and 10amino acids, 7 and 10 amino acids, 8 and 10 amino acids, 9 and 10 aminoacids, 5 and 9 amino acids, 6 and 9 amino acids, 7 and 9 amino acids, 8and 9 amino acids, 5 and 8 amino acids, 6 and 8 amino acids, 7 and 8amino acids, 5 and 7 amino acids, or 6 and 7 amino acids in length. Inspecific cases, the peptide is 5 amino acids in length, 6 amino acids inlength, 7 amino acids in length, 8 amino acids in length, 9 amino acidsin length, 10 amino acids in length, 11 amino acids in length, and soforth.

The peptide may comprise at least one of the 20 common amino acids innaturally synthesized proteins, or at least one modified or unusualamino acid, including but not limited to those shown on Table 1 below.

TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acid Abbr. AminoAcid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine Baad 3-Aminoadipicacid Hyl Hydroxylysine Bala (β-alanine, AHyl allo-Hydroxylysine(β-Amino-propionic acid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline4Abu 4-Aminobutyric acid, 4Hyp 4-Hydroxyproline piperidinic acid Acp6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid AIleallo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,sarcosine Baib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acidMeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelicacid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGlyN-Ethylglycine

In particular embodiments, the peptide may comprise some identity to,such as be derived from, a peptide of Table 2, and such a compositionmay be referred to a derivative of a peptide of Table 2. For example, apeptide that has one, two, or three amino acids that are different fromany of the peptides of Table 2 (including SPGARAF, DGPWRKM, and FGQKASS,for example) may be employed. Biologically functional equivalents of thepeptides of Table 2 may be employed and are thus defined herein as thosepeptides wherein selected amino acids may be substituted. Functionalactivity of these biologically functional equivalent peptides includesthe ability to home to dorsal root ganglion neurons, and this may betested in accordance with the exemplary methods of the inventionprovided herein.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and/or the like. Ananalysis of the size, shape and/or type of the amino acid side-chainsubstituents reveals that arginine, lysine and/or histidine are allpositively charged residues; that alanine, glycine and/or serine are alla similar size; and/or that phenylalanine, tryptophan and/or tyrosineall have a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and/or histidine; alanine, glycineand/or serine; and/or phenylalanine, tryptophan and/or tyrosine; aredefined herein as biologically functional equivalents.

To effect more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and/or chargecharacteristics, these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (0.4); threonine (0.7); serine (0.8);tryptophan (0.9); tyrosine (1.3); proline (1.6); histidine (3.2);glutamate (3.5); glutamine (3.5); aspartate (3.5); asparagine (3.5);lysine (3.9); and/or arginine (4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index and/or score and/or stillretain a similar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and/or those within ±0.5 are even moreparticularly preferred.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and/or antigenicity, i.e., with a biological property ofthe protein, for example. As detailed therein, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (0.4); proline (−0.5±1); alanine (0.5); histidine (0.5);cysteine (1.0); methionine (1.3); valine (1.5); leucine (1.8);isoleucine (1.8); tyrosine (2.3); phenylalanine (2.5); tryptophan (3.4).In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those which are within ±1 are particularly preferred, and/orthose within ±0.5 are even more particularly preferred.

In certain embodiments, the peptide is biocompatible that produces nosignificant untoward effects when applied to, or administered to, agiven organism according to the methods and amounts described herein.Organisms include, but are not limited to, mammals, including humans,dogs, cats, horses, cows, pigs, goats, and sheep. Such untoward orundesirable effects are those such as significant toxicity or adverseimmunological reactions. In preferred embodiments, biocompatiblepeptide-containing compositions will generally be peptides essentiallyfree from toxins, pathogens and harmful immunogens.

Peptides may be made by any technique known to those of skill in theart, including the expression of proteins and their further processingor the chemical synthesis of peptides, for example. In certainembodiments a peptide may be purified or isolated. Generally, “purified”will refer to a specific peptide composition that has been subjected tofractionation to remove various other proteins, polypeptides, orpeptides, and which composition substantially retains its activity, asmay be assessed, for example, by an assay as would be known to one ofordinary skill in the art for the specific or desired peptide.

III. Utilization of the Peptides of the Invention

In particular embodiments of the invention, one or more peptides fromTable 2, including SPGARAF, DGPWRKM, or FGQKASS, for example, areadministered to an individual in need thereof. In particular cases, theindividual has, is suspected of having, or is at risk for having one ormore peripheral neuropathies or neuropathic pain, for example.

Peripheral neuropathy as used herein refers to damage to the nerves ofthe peripheral nervous system, and this may result from diseases of thenerve or as a side effect of a systemic illness, for example. Peripheralneuropathies can present and have origin in a variety of ways, and insome cases the nerve is affected, whereas in other cases theneuromuscular junction is affected. Peripheral neuropathies may eitherbe symmetrical and generalized or focal and multifocal, and such apresentation is useful in determining the cause of the peripheral nervedisease.

Exemplary causes of peripheral neuropathy include, for example, geneticdiseases, such as Friedreich's ataxia or Charcot-Marie-Tooth syndrome,for example; metabolic/endocrine disorders, such as diabetes mellitus,chronic renal failure, porphyria, amyloidosis, liver failure, orhypothyroidism, for example; toxic origins, such as alcoholism, drugs(vincristine, phenyloin, isoniazid), organic metals, and/or heavymetals, for example; inflammatory diseases, such as Guillain-Barrésyndrome, systemic lupus erythematosis, leprosy, or Sjögren's syndrome;vitamin deficiency states, such as vitamin B12, vitamin A, vitamin E, orthiamin, for example; or other causes, such as malignant disease, HIV,radiation, or chemotherapy.

Generalized peripheral neuropathies are symmetrical, affecting theperipheral nervous system in its entirety. They may occur because of avariety of systematic illnesses and disease processes. These types ofneuropathies are further subdivided into several categories, includingdistal axonopathies, myelinopathies, or neuronopathies. Distalaxonopathies stem from some metabolic or toxic derangement of neurons,such as from metabolic diseases including diabetes, renal failure,deficiency syndromes (malnutrition or alcoholism, for example), or theeffects of toxins or drugs. Myelinopathies are the result of a primaryattack on myelin causing an acute failure of impulse conduction, such asfrom acute inflammatory demyelinating polyneuropathy (AIDP; akaGuillain-Barré syndrome), chronic inflammatory demyelinatingpolyneuropathy (CIDP), genetic metabolic disorders (e.g.,leukodystrophy), or toxins. Finally, neuronopathies are caused bydestruction of peripheral nervous system neurons, and they may be causedby motor neuron diseases, sensory neuronopathies (e.g., Herpes zoster),toxins or autonomic dysfunction, for example, although in some casesthey are caused by neurotoxins, such as vincristine, a chemotherapyagent.

The methods and compositions of the present invention may also beutilized for treating neuropathic pain. Neuropathic pain is chronic painthat often is accompanied by tissue injury, wherein the nerve fibersthemselves might be damaged, dysfunctional, or injured, for example.Such damaged nerve fibers send incorrect signals to other pain centers,therefore, the effect of a nerve fiber injury includes a change in nervefunction both at the site of injury and areas around the injury.

Neuropathic pain usually appears to have no obvious cause and oftenresponds poorly to conventional pain treatment, sometimes getting worseinstead of better with time. For some people, it can lead to seriousdisability. One exemplary type of neuropathic pain is phantom limbsyndrome, following removal of a limb. Other common causes ofneuropathic pain include alcoholism; back, leg, and hip problems;chemotherapy; diabetes; facial nerve problems; HIV infection or AIDS;multiple sclerosis; shingles; and/or spinal surgery, for example.Symptoms of neuropathic pain include numbness, shooting pain, burningpain, and/or tingling.

IV. Medical Conditions Affecting DRG Neurons

In particular embodiments of the invention, there are methods andcompositions suitable for use in the treatment of medical conditionsthat affect DRG neurons. In a specific embodiment, the medical conditionis a DRG neuronopathy.

In specific embodiments, the medical condition includes neuropathicpain, which can occur among patients with various metabolic andinflammatory disorders, including diabetic neuropathy for individualsthat suffer neuropathic pain caused by sensory neuron dysfunction. Inspecific embodiments, individuals with ganglionopathies, for example,caused by autoimmune diseases or drug toxicities, may utilize thepresent invention for neuropathic pain. In specific embodiments,treatment of these and other sensory neuronopathies by intrathecalDRG-targeting HDAd that express neurotrophic factors or analgesicpeptides is a useful application of the invention.

V. Therapeutic Proteins and Polynucleotides Encoding Same

In some embodiments, the present invention comprises therapeuticproteins, for example linked to DRG neuron-targeting peptide, orpolynucleotides encoding the therapeutic proteins. Exemplary therapeuticproteins include those that ameliorate at least in part a DRGneuronopathy or at least one clinical symptom caused directly orindirectly thereby. Exemplary agents include growth factors,transcriptional factors, signal peptides, or enzymes.

Exemplary genes and gene products that may be considered therapeutic incertain embodiments of the invention include the following: growthfactors such as NGF, EGF, FGF, BDNF, IGF1, CTNF, PDNF, or anti-apoptoticpeptides such as VAD, DEVD, or transcription factors such as NGN1, NGN2,NGN3, RUNX3, or signal peptides. Thus, in certain embodiments, NGF, EGF,FGF, BDNF, IGF1, CTNF, PDNF, VAD, DEVD, NGN1, NGN2, NGN3, RUNX3, IL-10,anti-TNFα, EPO, or prepro-β endorphin are employed.

Additional exemplary genes and/or proteins for use in the invention fortherapeutic benefit in conjunction with the peptide include thefollowing known in the art: self-complementary AAV8 via lumbar puncture(for example) (Storek et al., 2008); knockdown of Na_(v)1.3, Na_(v)1.8,VGCC, NMDA receptors and opioid receptors, spinal opioid receptors, P2Xreceptors and c-fos (Sinaiscalco et al. (2005); and NGF (including usingliposomes) (Wang et al., 2005). Additional references exhibiting use ofliposomes for peptide targeting include Du et al. (2007), McGuire et al.(2004), and U.S. Pat. No. 7,238,665, which is incorporated by referenceherein in its entirety.

VI. Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more peptides that target dorsal rootganglions and, in some cases, an additional agent dissolved or dispersedin a pharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one peptide will be known to those of skill in the artin light of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The peptide may utilize different types of carriers depending on whetherit is to be administered in solid, liquid or aerosol form, and whetherit need to be sterile for such routes of administration as injection.The present invention can be administered intravenously, intradermally,transdermally, intrathecally, intraarterially, intraperitoneally,intranasally, intravaginally, intrarectally, topically, intramuscularly,subcutaneously, mucosally, orally, topically, locally, inhalation (e.g.,aerosol inhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

The peptide may be formulated into a composition in a free base, neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a composition contained therein, its use inadministrable composition for use in practicing the methods of thepresent invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include one or more ofthe peptides and an aqueous solvent. As used herein, the term “lipid”will be defined to include any of a broad range of substances that ischaracteristically insoluble in water and extractable with an organicsolvent. This broad class of compounds are well known to those of skillin the art, and as the term “lipid” is used herein, it is not limited toany particular structure. Examples include compounds which containlong-chain aliphatic hydrocarbons and their derivatives. A lipid may benaturally occurring or synthetic (i.e., designed or produced by man).However, a lipid is usually a biological substance. Biological lipidsare well known in the art, and include for example, neutral fats,phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,glycosphingolipids, glycolipids, sulphatides, lipids with ether andester-linked fatty acids and polymerizable lipids, and combinationsthereof. Of course, compounds other than those specifically describedherein that are understood by one of skill in the art as lipids are alsoencompassed by the compositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the peptide may be dispersed in a solutioncontaining a lipid, dissolved with a lipid, emulsified with a lipid,mixed with a lipid, combined with a lipid, covalently bonded to a lipid,contained as a suspension in a lipid, contained or complexed with amicelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

A. Alimentary Compositions and Formulations

In preferred embodiments of the present invention, the peptide isformulated to be administered via an alimentary route. Alimentary routesinclude all possible routes of administration in which the compositionis in direct contact with the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered orally,buccally, rectally, or sublingually. As such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier,or they may be enclosed in hard- or soft-shell gelatin capsule, or theymay be compressed into tablets, or they may be incorporated directlywith the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792,451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

B. Parenteral Compositions and Formulations

In further embodiments, the peptide may be administered via a parenteralroute. As used herein, the term “parenteral” includes routes that bypassthe alimentary tract. Specifically, the pharmaceutical compositionsdisclosed herein may be administered for example, but not limited tointravenously, intradermally, intramuscularly, intraarterially,intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos.6,7537,514 6,737,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and5,399,363 (each specifically incorporated herein by reference in itsentirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in isotonic NaCl solution andeither added hypodermoclysis fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

C. Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the active compoundpeptide may be formulated for administration via various miscellaneousroutes, for example, topical (i.e., transdermal) administration, mucosaladministration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

VII. Kits of the Invention

Any of the peptide compositions described herein may be comprised in akit. In a non-limiting example, an additional agent may also becomprised in a kit. The kits will thus comprise, in suitable containermeans, a peptide and, in some cases, an additional agent of the presentinvention.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there are more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present invention also will typically include a meansfor containing the peptide and any other reagent containers in closeconfinement for commercial sale. Such containers may include injectionor blow molded plastic containers into which the desired vials areretained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The compositions may alsobe formulated into a syringeable composition, in which case thecontainer means may itself be a syringe, pipette, and/or other such likeapparatus from which the formulation may be applied to an infected areaof the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Isolation of DRG Neuron-Binding Peptides

Adult C57BL/6 mice aged 8-12 weeks were used in all experiments, andwere housed in an animal room with a 12-h light and 12-h dark cycle inan illumination-controlled facility. All experiments were conducted withthe approval of the Research Center for Animal Life Science at ShigaUniversity of Medical Science. A phage library expressing random 7-merpeptides fused to a minor coat protein (pill) of the M 13 phage, at acomplexity of about 1.3×10⁹ independent sequences, was purchased fromNew England BioLabs (The Ph.D.-C7C Phage display Peptide Library kit,Beverly, Mass.). Rabbit anti-fd bacteriophage antibody was purchasedfrom Sigma Aldrich Corp. (St. Louis, Mo.). Donkey FITC-labeledanti-rabbit IgG was purchased from Chemicon (Temecula, Calif.). Thethree synthetic peptides, DRG1: SPGARAF (SEQ ID NO:1), DRG2: DGPWRKM(SEQ ID NO:2), DRG3: FGQKASS (SEQ ID NO:3), were supplied from YanaiharaInstitute (Shizuoka, Japan). Various cell lines; mouse neuroblastomacells (Neuro-2a), human embryonic kidney epithelial cells (HEK-293),opossum kidney cells, were purchased from American Type CultureCollection (Manassas, Va.) for use in a subtractive panning protocol.These cultured cells were maintained in DMEM medium with 10% fetal calfserum (FCS, Invitrogen, Gaithersburg, Md.) with antibiotics at 37° C. in5% CO₂.

Mouse DRG neurons were isolated from lumbar segments L1-L5 of C57BL/6mice using an enzymatic procedure described previously (Terashima etal., 2005). The neurons were purified with 30% percoll (MP Biomedicals,Solon, Ohio), washed twice with Ham's F12 medium supplemented with 10%fetal calf serum (Invitrogen), and then plated on poly-L-lysine(Sigma)-coated 10 cm dishes for in vitro biopanning or on 8 well Lab-TekChamber Slides (NUNC, Naperville, Ill.) for in vitro phage binding. Thecells were allowed to attach at 37° C. for 12 hours in 5% CO₂.

The in vitro phage display library screening protocols described by Honget al. (2000) were employed with minor modifications. Briefly, afterwashing isolated DRG neurons three times with PBS, the culture mediumwas changed FCS-free DMEM medium. Then, 1×10¹⁰ plaque-forming units(pfu) of the M13 phage library were added to isolated DRG neurons(1×10⁶/well), and incubated at 37° C. in 5% CO₂ for 10 min. Afterwashing three times, the bound phages were recovered by grinding thecells with protease inhibitor in DMEM medium. Aliquots were dilutedappropriately and infected E. coli ER2738, mixed with Agarose Top, andincubated in LB/IPTG/X-gal plates at 37° C. overnight. After titratingplaque forming units, phages in the aliquot were further amplified byinfecting E. coli with them in a shaker at 37° C. for 4.5 h. Phages fromthe E. coli were purified, and 1×10¹⁰⁻¹¹ pfu were added to isolated DRGneurons again. In all, five such rounds of screening with DRG neuronswere carried out. To select more specific phages for DRG neurons, asubtractive panning protocol (Nicklin et al., 2000) against irrelevantcell lines (Neuro-2a, HEK-293, and opossum kidney cells from AmericanType Culture Collection, Manassas, Va.) was performed twice. Thesecultured cells were maintained in DMEM medium with 10% fetal calf serum(FCS, Invitrogen, Gaithersburg, Md.) with antibiotics at 37° C. in 5%CO₂. To avoid the selection of polystyrene and poly-L-lysine-specificpeptides, a subtractive panning protocol against poly-L-lysine culturedishes was also performed twice (Adey et al., 1995).

In vitro phage binding to DRG neurons was carried out on culturedneurons in 8-well chamber slides following three washes with PBS toclear the cell surface (n=1×10⁵ cells/well). Cells were incubated at 37°C. for 1 h with either the three selected phages or control libraryphage at 2×10⁹ pfu/well (1×10¹⁰ pfu/ml), with or without syntheticpeptides (10⁻⁴ or 10⁻⁶ M; DRG1: SPGARAF (SEQ ID NO:1), DRG2: DGPWRKM(SEQ ID NO:2), DRG3: FGQKASS (SEQ ID NO:3)), which had 7 amino-acidsequences homologous to the selected phages. Subsequently, unboundedphages and/or peptides were removed by washing the chamber slides threetimes with PBS. To detect phages bound to the cells, the cultured cellswere fixed with 4% paraformaldehyde and incubated for 1 h with rabbitanti-fd antibody (diluted 1:1000 in PBS with 3% donkey serum, SigmaAldrich Corp., St. Louis, Mo.), washed three times with PBS, andsubsequently incubated with FITC-conjugated donkey anti-rabbit IgGdiluted 1:1000 for 1 h (Chemicon, Temecula, Calif.). After three washes,the cells were treated with propidium iodine (PI) for nuclear staining,and then observed under a confocal laser-scanning microscope (LSM510,Carl Zeiss, Jena, Germany). For quantitative analysis, ten randomlyselected fields in each well were photographed and diameters weremeasured (long and short) of the cells positive for phage immunostainingand PI staining. The mean cell size was calculated as the mean of thelong and short diameter, and cell-size differences were evaluated indifferent groups.

To evaluate their binding to DRG cells in vivo, GST fusion proteins ofthree selected peptides were synthesized using the pGEX4T-1 vector kit(Amersham, Piscataway, N.J.) according to the manufacturer'sinstructions. Briefly, oligonucleotides for C-DRG1-C, C-DRG2-C, andC-DRG3-C were synthesized cloned into the pGEX4T-1 vector, andtransformed in E. coli. The synthesized GST-fusion proteins were pulldown by Glutathione Sepharose 4B (Amersham), and purified. The puritiesand reactions to anti-GST antibodies (Amersham) were confirmed bygel-electrophoresis stained by Coomassie Blue and anti-GSTimmunoblotting.

Under intraperitoneal pentobarbiturate anesthesia (0.1 mg/g BW), 10 μl(500 μg/ml) of purified GST-fusion proteins (GST-DRG1, GST-DRG2,GST-DRG3, or GST alone) were injected into the subarachnoid space of8-week-old female C57BL/6 mice (n=3 each). After 1 h, the mice weresacrificed and transcardially perfused with cold saline followed by 4%paraformaldehyde, 0.5% glutaraldehyde and 0.3% picric acid in 0.1 M PBS(pH 7.4). The L5 DRGs were then isolated and cut sectioned (10 μm) forfluorescence immunohistochemistry. After blocking with 10% horse serum,the sections were incubated overnight at 4° C. with goat anti-GSTantibody (Amersham) diluted 1:1000 and rabbit anti-neurofilament L(NF-L, non-phosphorylated form, Chemicon, Temecula, Calif.) antibodydiluted 1:100 in PBST. The sections were incubated for 1 h with AlexaFluor 488-labeled anti-goat IgG and Alexa Fluor 555-labeled anti-rabbitIgG (Molecular Probes, Eugene, Oreg.) at room temperature, mounted withVECTASHIELD mounting medium containing 4′,6-diamidino-2-phenylindole(DAPI) (Vector Laboratories Inc., Burlingame, Calif.), and observedunder a fluorescence microscope (Axioplan 2 imaging and Axiocam, CarlZeiss, Jena, Germany). For quantitative analysis, the inventors tookpictures of randomly selected fields and measured diameters (long andshort) of the cells positive for GST, NF-L and DAPI. The mean cell sizewas calculated as the mean of the long and short diameters, andcell-size differences were evaluated in the three different groups.

BLAST (see the National Center for Biotechnology Information's website)searches of the mouse database with the phage peptide sequences werecarried out to identify homologies with proteins of interest, includingneuronal growth factors, cytokines, hormones, and cell adhesionmolecules.

Data were analyzed by ANOVA for multiple comparisons and shown asmean±SD. Significance was assigned at P<0.05.

The inventors used an in vitro biopanning protocol and isolated 113phage plaques in five rounds of screening with DRG neurons and fourrounds of negative selection, two against irrelevant cell lines, and twoagainst poly-L-lysine-coated culture plates. Of the 113 plaques, fivedisplayed an identical 7-amino acid sequence (DRG1), four shared another7-mer sequence (DRG2), and three others displayed a third sharedsequence (DRG3); 17 other sequences (DRG4 to DRG20) were each shared bytwo different plaques. The sequences of the other 67 clones were alldifferent from one another (Table 2).

Name Sequence Counts SEQ ID NO DRG1 SPGARAF 5  1 DRG2 DGPWRKM 4  2 DRG3FGQKASS 3  3 DRG4 TGFQSGS 2  4 DRG5 DSSRTRL 2  5 DRG6 DFIRTQA 2  6 DRG7LKHINEA 2  7 DRG8 GAHNNN 2  8 DRG9 NPHKAPN 2  9 DRG10 NPSLQAP 2 10 DRG11PPWSSPK 2 11 DRG12 AQSHNKL 2 12 DRG13 LPTSKKM 2 13 DRG14 NHLKNPA 2 14DRG15 TFSIGEK 2 15 DRG16 QAIQNST 2 16 DRG17 HNTNAQH 2 17 DRG18 TPSLPQT 218 DRG19 NMPTQRS 2 19 DRG20 PVRSPAV 2 20 DRG21 SSQAPQS 1 21 DRG22DAQKNMN 1 22 DRG23 GLQLSQT 1 23 DRG24 SASNTQY 1 24 DRG25 EGHLVSQ 1 25DRG26 SDPGNYM 1 26 DRG27 ALDNVPH 1 27 DRG28 PTKQHAK 1 28 DRG29 TELQRHN 129 DRG30 HTTSSLY 1 30 DRG31 MGQNLRF 1 31 DRG32 NLQLAPD 1 32 DRG33SSFRGAT 1 33 DRG34 LHKSALL 1 34 DRG35 APPELRL 1 35 DRG36 HRTIASG 1 36DRG37 TESIGDK 1 37 DRG38 APDETER 1 38 DRG39 KGLPPGH 1 39 DRG40 PSGTPSY 140 DRG41 SNRSPLM 1 41 DRG42 TIGQSYR 1 42 DRG43 SPTEGTP 1 43 DRG44PLSGAPW 1 44 DRG45 DAPTHMH 1 45 DRG46 TDFRSRV 1 46 DRG47 LVLPPLA 1 47DRG48 SSSPARL 1 48 DRG49 TATNTRT 1 49 DRG50 DGAGTWV 1 50 DRG51 EKHLAPR 151 DRG52 PLTPLGF 1 52 DRG53 MTPFMGS 1 53 DRG54 DSPGWPH 1 54 DRG55GERHSLT 1 55 DRG56 TTAVALR 1 56 DRG57 NGLHVQR 1 57 DRG58 TLSPRSA 1 58DRG59 HTGPFGL 1 59 DRG60 LSTSSKK 1 60 DRG61 TPPSPRT 1 61 DRG62 PALSHST 162 DRG63 TPSWSKK 1 63 DRG64 STPAVPP 1 64 DRG65 NLNAHHK 1 65 DRG66QHQKQGY 1 66 DRG67 NKTTNIM 1 67 DRG68 TSASLSS 1 68 DRG69 RSSPPNT 1 69DRG70 SPPRPTG 1 70 DRG71 VNTPERH 1 71 DRG72 TPQYPKL 1 72 DRG73 PTLLPHQ 173 DRG74 NNANYRL 1 74 DRG75 GPHFHQS 1 75 DRG76 PAMNSVK 1 76 DRG77GTTPTST 1 77 DRG78 HNSTRGS 1 78 DRG79 DDSGPLR 1 79 DRG80 NMHPTAT 1 80DRG81 HQNWRHT 1 81 DRG82 PSTKYHS 1 82 DRG83 PLRLAHQ 1 83 DRG84 QMPGNNL 184 DRG85 LATPLRN 1 85 DRG86 LNGLKAA 1 86 DRG87 TVSSHRA 1 87

The three phage clones, DRG1, DRG2 and DRG3, were further analyzed sincethese 3 clones appeared at least three times by binding to DRG neuronsin vitro; these phage clones were designated DRG-p1, DRG-p2 and DRG-p3,respectively.

The binding of the 3 different clones to isolated DRG neurons maintainedin culture was analyzed (FIG. 1). DRG-p1 bound to 38% of the DRG neurons(83/219), DRG-p2 to 43% (100/231), and DRG-p3 to 29% (69/235), while thecontrol phage library bound to 8% (45/539) of the DRG neurons. Theaddition of peptides (10⁻⁴ M) with identical amino-acid sequencesblocked the staining with anti-phage antibodies by 97% for DRG-p1, 94%for DRG-p2, and 94% for DRG-p3, while adding 10-6 M peptide blockedstaining by 47% for DRG-p1, 54% for DRG-p2, and 53% for DRG-p3, ascompared with similarly bound cultures stained with antibody alone. Aseach peptide was only capable of competing for the binding of the phagewith an identical 7-amino acid sequence (data not shown), thephage-neuron binding was concluded to be sequence specific (FIG. 1B).

The cell size distribution of the DRG neurons that bound to individualphage clones was then analyzed. Interestingly, DRG-p1 and DRG-p3staining occurred mainly in small-sized neurons with a mean diameter of22.0±5.3 μm (for DRG-p1) and 23.2±6.7 μm (for DRG-3p), while DRG-p2staining occurred mainly in large-sized neurons with a mean diameter of30.9±7.0 μm (FIG. 1A). There was a significant difference between themean diameter of the cells positive for DRG-p1 and that for DRG-p2(p<0.001), and of the cells positive for DRG-p2 and for DRG-p3(p<0.001), but no difference between the cells positive for DRG-p1 andDRG-P3.

To examine the DRG-targeting of peptides in vivo, individualGST-peptide-fusion proteins (5 μg each) or GST alone were injected intothe subarachnoid space in C57BL/6 mice, the DRGs were isolated, and theywere used in anti-GST immunostaining to detect the neuron-associatedGST-peptide-fusion proteins (see Materials and Methods). Additionally,the inventors double-immunostained the cells against anti-neurofilamentL (NF-L, non-phosphorylated form) antibodies, and found that essentiallyall (99%) cells positive for GST-peptide-fusion proteins were alsopositive for neurofilament (FIG. 2B). In DRG cells from control miceinjected with GST, no anti-GST staining was observed (FIG. 2B).Furthermore, 39% (368/949) of the NF-L-positive neurons were positivefor GST-DRG1, 25% (214/848) for GST-DRG2, and 14% (147/1081) forGST-DRG3. Cell size analysis showed that GST-DRG1- and GST-DRG3-positivecells were mainly small-sized neurons, whereas GST-DRG2-positive cellswere mainly large-sized DRG neurons (FIG. 2B). The mean diameter of thecells positive for GST-DRG1 was 21.6±5.2 GST-DRG2 33.5±7.4 μm, andGST-DRG3 22.7±6.6 μm. There was a significant difference in meandiameter between GST-DRG1-positive cells and GST-DRG2-positive cells(p<0.001), and between GST-DRG2-positive and GST-DRG3-positive cells(p<0.001), but no size difference between GST-DRG1-positive andGST-DRG3-positive cells. Therefore, the cell size specificity of thethree different peptides in phages in vitro (FIG. 1A) was confirmed bythe in vivo-binding experiment. Moreover, as most of the GST-positiveimmunoreactivity was detected in the cytoplasm of the DRG neurons (FIG.2B), and as anti-GST antibodies used in this experiment were specificfor recombinant GST-peptides fusion proteins synthesized in E. coli, anddid not react with mouse endogenous GST, the bound GST-peptide-fusionproteins appear to have been internalized into the cytosol of thetargeted neurons. In contrast, the GST immunoreactivities in the centralnervous system (brain and spinal cord) was examined, and there was nospecific staining. Possible endogenous candidates in the mouse proteindatabase were searched for that might bind to the DRG, “receptors” forthe three 7-amino acid peptides, and the same DRG1 7-amino-acid sequencein the extra cellular matrix protein TM14 (NP_(—)077199.2) was found (deVega et al., 2007). However, 6 out the 7 amino acids were located in theputative signal peptide and not in the putative binding domain in TM14.The mouse neuronal differentiation-related protein (NDRP,NP_(—)001096649.1) has the same 7-amino acid sequence as that in DRG2(Kato et al., 2000). NDRP is expressed in the sensory neuronal systemincluding in DRG neurons, though the current knowledge of its functionis limited. There were no peptide sequences in the database similar tothat in DRG3.

The inventors used the in vitro screening of a C7C peptide librarydisplayed on M13 filamentous phages against isolated mouse DRG neuronsto isolate phage-clones that encompassed three different 7-amino acidpeptides that displayed specific binding for these neurons in vitro.Immunohistochemical analysis following the injection of GST-peptidefusion proteins in vivo demonstrated that the three peptides bind to DRGneurons with different sizes; furthermore, these fusion proteins wereinternalized into the cytoplasm of the targeted neurons.

The Ph.D.-C7C Phage display Peptide Library kit expresses random 7-aminoacid peptides fused to the minor coat protein (pIII) of the M13 phage.By sequence analysis, only 113 phage plaques were represented in thebound peptide, consistent with a very efficient selection process (Table2). The final binding studies allowed identification of three phages(DRG1, DRG2, or DRG3) that occurred repeatedly. In specific embodimentsof the invention, the efficient identification of these clones was theresult of multiple rounds of positive and negative selection using thebiopanning protocol (Liang et al., 2006), although in specific casessome of the other 84 clones (Table 2) are true DRG-bindingpeptide-phages. The three clones that were analyzed further showedinteresting specificities to the targeted neurons: DRG-p1 and DRG-p3 tosmall-sized neurons and DRG-p2 to large-sized neurons. Moreover, thesize specificity of target neurons for individual peptides was confirmedby in vivo binding experiments: GST-DRG1 and GST-DRG3 recognizedsmall-sized neurons, and GST-DRG2 recognized large-sized neurons. Sinceboth DRG1 and DRG3 recognized small sized neurons, two peptide ligandsmight bind to the same cell populations. Therefore, double staining wasperformed of isolated DRG neurons after the incubation with DRG-p1 andGST-DRG3 by antibodies against phage and GST, respectively. Doublepositive staining was obtained in 51% of DRG-p1-positive neurons, andwas in 71% of GST-DRG3-positive neurons, respectively. The resultindicates that both DRG1 and DRG3 recognized different targetingmolecules, but may bind to the same cell populations in specificsubpopulations in small-sized neurons. Histologically, mammalian sensoryDRG neurons have been classified into two major types: large-light andsmall-dark cells on the basis of their staining characteristics seenunder light microscopy (Lawson, 2002). Clinically, peripheral sensoryneuropathy (including neuronopathy) has been classified into two typesbased on fiber size: large fiber sensory neuropathy and small fibersensory neuropathy (Mendell and Sahennk, 2003; Sghirlanzoni et al.,2005). The symptom typically found in large fiber sensory neuropathy isan ataxic gait, and that in small fiber sensory neuropathy is mainlypain (Mendell and Sahennk, 2003; Sghirlanzoni et al., 2005). Neuronalsize is directly related to axonal characteristics (Lawson, 2002; Lawsonand Waddell, 1991; Sghirlanzoni et al., 2005). Thus, each targetingmotif included in DRG1, DRG2, DRG3, and, potentially, in some of theother 84 peptides sequences, may be useful tools for pathophysiologicalanalysis such as a cell-identification marker, or for the generation ofgene-delivery constructs for sensory neuropathies (Liu et al., 2005;Mata et al., 2006; Sah, 2006).

The inventors also tried to determine the size of the targeted proteinusing the whole homogenate of DRG tissues from mice, and found somespecific bands by Western blotting analysis. Since those bands areproteins targeted by the peptides, in specific aspects of the invention,they are further clarified.

In conclusion, the inventors have used in vitro phage display technologyto isolate and identify three different exemplary peptides that bind toDRG neurons in vitro and in vivo. It was further shown that the peptidesrecognize neurons of two different sizes. In further studies the bindingproteins and their receptors are identified. Nevertheless, the three DRGneuron-specific peptides are useful for the generation of moleculardelivery systems targeting different DRG neurons in vivo.

Example 2 Production of Fiber-Modified Helper Virus

In a routine method for HDAd production, all necessary factors forpackaging HDAd genome are supplied in trans by a helper virus (HV), afirst generation Ad. In this scheme, HDAd that expresses the gene ofinterest (e.g., a therapeutic transgene) can be made to target specificorgan or cell type simply by using an appropriate HV at the finalamplification (Kim et al., 2001). There is no need to engineer a newHDAd (FIG. 3), in specific embodiments. To produce targeted HV, theinventors first attenuated the natural tropism by ablating the bindingsites of Ad5 fiber to its canonical primary docking receptors, CAR andHSPG (Kritz et al., 2007). To circumvent the very poor infectivity ofKO1S* mutant Ad (which lack both binding sites) toward 293 cells, 293cells were established expressing Ad5 wild type fiber (293-fiber) toincorporate wild type fiber into the capsid of HV-KO1S* (FIG. 3 a).Fiber-modified HV generated on 293-fiber had both wild type fiber andmodified fiber encoded by HV genome. A total of 5 HVs were generated:[i] HV-WF containing wild type fiber: [ii] HV-KO1S* a detargeted HV; and[iii, iv, & v] 3 HVs on a HV-KO1S* backbone that contains individualDRG-targeting motifs, DRG1, DRG2 and DRG3 (FIG. 4 a) (Oi et al., 2008).Fiber-modified HVs were initially generated on 293-fiber cells andsubsequently amplified on 293 cells to remove wild type fibers in thefinal HV capsid. Proper fiber modification in the genome of individualHVs was verified by DNA sequencing (FIG. 5 b).

Example 3 HV Containing DRG Neuron Targeting Motifs Efficiently andSpecifically Transduces DRG Neurons In Vitro and In Vivo

As compared to wild-type fiber HV (HV-WF), HV-KO1S* showed 100-foldlower infectivity to 293 cells (FIG. 4 b, note log scale), indicatingthat HV-KO1S* had lost its capacity to interact with primary dockingsites on 293 cells. As the different DRG motif-containing HVs wereengineered on an HV-KO1S* backbone, they also transduced 293 cellspoorly (FIG. 4 b, see also FIG. 6 a). Transduction of isolated DRGneurons by HV-WF was about 100-fold less efficient than that of 293cells. In contrast, HVs containing DRG targeting motifs 1-3 alldisplayed markedly enhanced transduction of isolated DRG neurons (FIG. 4c, note log scale). Interestingly, HV-DRG1 showed the highesttransduction efficiency followed by DRG2 and DRG3. Compared to HV-WF,HV-DRG1 showed 100-fold higher efficiency of transduction of DRG neuronsin vitro, while HV-KO1S* displayed minimal transduction capacity (<1% ofthat of HV-WF, and <0.001% that of HV-DRG1). (see also FIG. 6 b).

The transduction efficiency of DRG neurons was investigated by thedifferent HVs in vivo by injecting 1×10⁸ viral particles (vp) of HVscontaining dual βgeo (β-galactosidase-neo fusion protein) and luciferasegene cassettes into the subarachnoid space of C57BL/6 mice and comparedthe biodistribution of the-fiber modified HVs five days later. X-galstaining was barely detectable in DRG of mice treated with HV-WF orHV-KO1S*. In contrast, transduction of DRG by HVs containing DRGtargeting motifs was evident from the X-gal staining (FIG. 4 d, FIG. 7c). Occasional punctate X-gal reaction product was detected in the rootfibers leading to the DRG in HV-WF treated mice. Close microscopicobservation revealed rare X-gal positive areas in the brain of micetreated with HV-WF (e.g., an X-gal positive area was identified in thelateral ventricular region. see FIG. 7 a online), but almost never inthe brain of mice treated with other HVs. The spinal cord stainedpositive around the vector injection site and at the dorsal intermediatesulcus and central canal in mice treated with HV-WF. Although scantstaining was found in the injection sites of mice treated with HV-DRG1,-DRG2 and -DRG3, there was no staining in mice treated with HV-KO1S*(FIG. 4 d, FIG. 7 b). X-gal positive areas were also identified on thesurface of the sciatic nerve in the WF, and to a much smaller extent,the DRG1, DRG2 and DRG3 groups, but not in the KO1S* group (FIG. 7 d).

The observation based on X-gal staining pattern was corroborated byquantifying HV-induced luciferase activity. Again, there was lowluciferase activity in the HV-WF group in all tissues, whereas highluciferase activity was present almost exclusively in the DRG of thethree DRG-targeting HV-treated animals. Transduction by HV-DRG1 and DRG2was 100-fold higher than that by HV-WF. HV-DRG3 was substantially lessefficient, being only 4-fold higher than HV-WF (FIG. 4 e, note logscale). Luciferase activity from HV-KO1S* (detargeted) was negligible inall tissues. Long-term LacZ mRNA expression induced by HV-WF and HV-DRG1by quantitative RT-PCR. The expression of LacZ mRNA decreased over timein both groups (FIG. 4 f, note log scale). By 60 days, LacZ mRNA wasbarely detectable in the WF group, but was still present at asubstantial level in the DRG1 group.

Example 4 Fiber-Modified HDAd Vectors Expressing Cre Turn on GFPExpression in DRG Neurons of Rosa-GFP Mice

To test whether HDAd containing DRG targeting motifs in the fibertransduce the intended targets, ROSA-GFP mice were utilized, in whichtransgenic GFP expression is interrupted by the Stop fragment flanked byloxP sequences. Upon Cre-mediated excision of the Stop fragment, GFP isexpressed (FIG. 8 a). HDAd expressing Cre driven by EF-1 promoter weregenerated using HV-WT (HDAd-WF-Cre) and then the inventors switchedvector capsids by coinfection with HDAd-WF-Cre and HV-DRG 1, -DRG2,-DRG3 or -KO1S* (FIG. 3 c). HDAd were injected locally into thesubarachnoid space, mice were sacrificed 5 days later, and mRNAexpression was determined by real time RT-PCR. The expression of Cre andGFP in different tissues (FIGS. 8 b,c) was consistent with thebiodistribution of HV (FIG. 4 c-e). All DRG neuron-targeting HDAdexpressed Cre and GFP 10-50 fold higher than HDAd-WF-Cre in DRG, whileHDAd-KO1S* showed minimal expression. The Cre mRNA level was barelydetectable in brain and sciatic nerve. It was generally low in spinalcord, in which tissue the HDAd-WF-Cre led to the highest expressioncompared with the other tissues and other vectors tested (FIG. 8 b). GFPmRNA expression was barely detectable in all three sites, i.e., brain,spinal cord and sciatic nerve (FIG. 8 c). Furthermore, very low levelsof Cre and GFP mRNA were detected in DRGs of the HDAd-WF-Cre-treatedgroup (FIGS. 8 b,c). In contrast, HDAd-DRG1, -DRG2 and -DRG3 led toparallel high level expression of both Cre and GFP mRNA expression inDRGs of injected animals. In all cases, HDAd-KO1S* induced Cre and GFPexpression was essentially undetectable. The relative mRNA levels werealso reflected by the relative expression of Cre and GFP proteins byimmunoblot analysis (FIG. 8 d). HDAd-DRG1-Cre and -DRG2-Cre induced30-fold higher protein levels than HDAd-WF-Cre. Similarly, GFPexpression in mice treated with HDAd-DRG1-Cre or -DRG2-Cre was over100-fold higher than that with HDAd-WF-Cre, while it was virtuallyundetectable in mice treated with HDAd-KO1S*-Cre (FIG. 8 d).

By fluorescent microscopy, GFP fluorecence was readily detected inindividual neurons of DRGs transduced by HDAd-DRG1 and -DRG2 (FIG. 8 e,top panel). HDAd-DRG3-transduced DRGs emitted weak fluorescence; HDAd-WFalso produced detectable but even weaker fluorescence. No fluorescencewas detected in the KO1S* group. The proportion of GFP+ cells to DAPI+neurons was 88.1% in the DRG1 group, 67.7% in the DRG2 group, 16.9% inthe DRG3 group, and 6.3% in the WF group, respectively (FIG. 8 e, bottompanel). A few scattered GFP+ areas were found in the brain and sciaticnerve of the WF group (Arrows, FIG. 9), while GFP+ areas were identifiedin the spinal cord at or close to the injection sites in all except theKO1S* group (FIG. 9).

Example 5 HDAds Produce Long-Term Transgene Expression with MarkedlyAttenuated Inflammatory Response

Transgenes delivered by Ad including HDAd are essentially not integratedinto the host genome, and their expression goes down with cell turnover.The level of Cre and GFP mRNA expression was compared in the DRG ofHDAd-DRG1 and HDAd-WF treated groups. HDAd-WF treatment led to low levelexpression of both at day 5 with rapid decline in relative expressionover the next two months, such that only 22.9% (for Cre) and 14.5% (forGFP) of the initial (5 day) mRNA level remained at 60 days (FIGS. 8f,g). In contrast, HDAd-DRG1 treated DRGs led to greatlyenhanced >100-fold higher Cre and GFP expression at day 5. Over the twomonths afterwards, mRNA expression went down at a greatly reduced rateand was 23.3% for Cre and 18.5% for GFP mRNA at day 60.

To further examine both the long-term transgene expression by HDAd andits effect on the host inflammatory response, βgeo expressing HDAd andHV were injected into mice. The inventors first quantitated LacZ mRNAexpression for the next two months (FIG. 10 a). Arbitrarily setting theLacZ mRNA expression level at day 5 as 100%, in the HDAd groups (forboth HDAd-WF or HDAd-DRG1), expression level gradually decreased to ˜20%at day 60. When the corresponding HVs (HV-WF or HV-DRG1) were used, thedecline in mRNA was substantially faster, going down to about 0.4-1.7%of the initial level at day 60. Thus, the relative stability oftransgene expression depends on the vector (HV vs HDAd), lastingsubstantially longer for HDAd than for HV, and not on the fiber type(wild-type, WF, vs DRG-targeted, DRG1). However, since the HDAd-DRG1 ledto 100-fold higher initial expression, at the end of 60 days,HDAd-DRG1-mediated LacZ expression was still readily apparent whileHV-mediated expression was no longer evident.

HDAd does not produce any viral proteins and HDAd-mediated transgeneexpression is largely free of chronic inflammatory response.Nonetheless, an acute inflammatory response can happen following HDAd orHV administration as a result of innate immunity. Acute toxicity wasmonitored by measuring cytokines secreted into the CSF. IL-6 level wasincreased 3 hours after vector injection in all groups, but to a muchlesser extent in mice treated with HDAd. However, IL-6 productionfollowing HDAd-KO1S* and HDAd-DRG1 was significantly lower than thatafter HDAd-WF injection (FIG. 10 b). Although IL-6 was still elevated inmice treated with HV-WF 24 hours later, it returned to the level similarto that treated with PBS (FIG. 11 a) in all other groups, suggestingthat attenuation of natural tropism reduces Ad-associated induction ofIL-6. The secretion of TNF-α was not influenced by detargeting (FIG. 10c, FIG. 11 b). However, the response to HDAd and HV differed markedly.HV produced a robust TNF-α response that lasted at least two weeks,while the response to HDAd was greatly attenuated (FIG. 10 c). Thekinetics of CSF IL-1β differed from that of IL-6 or TNF-α (FIG. 10 d).Importantly, PBS alone increased IL-1β 4-fold 3 hours after injectionand returned to pretreatment level by day 3 (FIG. 11 c). IL-113 levelwas elevated in all groups 3 hours after the vector injection. Althoughthe increase of IL-1β in the HV groups was higher than the HDAd groups,the difference, especially early on (at 3 h), was much smaller incomparison to that observed for IL-6 or TNF-α.

Example 6 A Single Injection of DRG-Targeting HDAd Expressingβ-Hexosaminidase Restores Enzyme Activity and Function of SensoryNeurons in a Mouse Model of Sandhoff Disease

DRG neurons are involved in many neurological diseases which impair thestructural integrity and function of sensory neurons. However, few wellcharacterized animal models are available to test the effects ofrestoration of function in sensory neurons. A mouse counterpart ofSandhoff disease in humans was a useful model for this purpose. Sandhoffdisease is caused by mutations in the β-hexosaminidase β-subunit gene.Absence of this enzyme subunit leads to defective GM2 gangliosidedegradation and massive accumulation of the ganglioside and othersubstrates in the lysosomes of neurons. The mouse model for thisdisease, hex^(−/−) mice, has been shown to develop progressiveimpairment of motor function (Sango et al., 1995). The DRG of these miceexhibit the typical histopathological changes of GM2 gangliosidosis;furthermore, the animals also display abnormal sensory nerve conductionvelocity and action potential, objective measurements that can be usedto gauge the severity of the disease.

The effect of DRG neuron-targeted enzyme replacement was investigated onthe abnormal histology and sensory neuronal impairment in hexb^(−/−)mice. The inventors injected 1×10⁸ vp of HDAd vectors expressing hexbdriven by the EF-1 promoter into the subarachnoid space of hexb^(−/−)mice at 4 weeks of age and measured mRNA level and enzyme activity innerve tissues 8 weeks later. An empty vector was used as control. Inhexb^(+/+) mice, all neural tissues expressed hexb mRNA, emptyvector-treated hexb^(−/−) mice displayed no detectable hexb mRNA, whilehexb^(−/−) mice treated with HDAd-DRG1-hexb (DRG1-hexb) expressed hexbmRNA almost exclusively in DRG, at a level that was significantly higherthan wild type mice (FIG. 12 a). The wild type fiber HDAd (WF-hexb)produced hexb mRNA in brain and spinal cord at a level that wassubstantially lower than that in wild type mice. Hex A is a heterodimerof α and β subunits and Hex B is a homodimer of hexosaminidase βsubunits. Expression of hexb rescues both Hex A and Hex B activities inhexb^(−/−) mice. After treatment, total Hex and Hex A activities in DRGincreased to 75-76% of those in wild type mice (FIGS. 12 b,c), and Hex Bactivity was similar to that in wild type mice (FIG. 12 d). The smalldiscrepancies between mRNA level and enzyme activities were probablyattributed to the suboptimal ratio of the two subunits. As in wild typemice, hexb protein expression in DRG neurons in DRG1-hexb-treatedhexb^(−/−) mice was confirmed by immunofluorescence, which showed thatmost DRG neurons were positive for Hexb (FIGS. 12 f,h). In contrast,immunofluorescence staining was totally absent in the empty vector groupand barely detectable in the WF-hexb-treated DRG (FIGS. 12 e,g).Histochemically, Hex enzyme activity was revealed in the section in situby a purple-brown coloration resulting from diazonium coupling betweenfast Garnet GBC and the products catalyzed fromnaphtol-AS-BI-D-N-acetyl-β-glucosaminide by hexosaminidase. In agreementwith enzyme activity, the histochemical stain in DRG1-hexb treated micewas present at a level similar to that in hexb^(+/+) mice, while micetreated with WF-hexb exhibited much lower coupling and those with theempty vector displayed no evident coupling (FIG. 12 i-1). Sandhoffdisease is a serious gangliosidosis that affects both the central andthe peripheral nervous systems and is associated with a much shortenedlifespan (mice at 16-20 weeks of age had to be euthanized because ofsevere debilitating motor dysfunction.) DRG1-hexb treatment did notextend the time until euthanasia. However, progression of objectivemeasures of DRG function was markedly improved by the treatment. TheDRG1-hexb group maintained intact sensory nerve conduction velocity(SNCV) at a normal level 4 and 8 weeks after treatment, while micetreated with empty or WF-hexb group exhibited significantly impairedSNCV (FIG. 13 a). Unlike the intact SNCV at 8 weeks in mice treated withDRG1-hexb, the motor nerve conduction velocity (MNCV) deteriorated atsimilar rates in all treatment groups compared to untreated wild typecontrols (FIG. 14 a). Sensory nerve action potential (SNAP) deterioratedmarkedly in empty vector-treated hexb^(−/−) mice at 8 weeks aftertreatment (12 weeks of age). DRG1-hexb treatment group maintained theirSNAP at the same level between 4 and 8 weeks, whereas WF-hexb-treatmentfailed to slow down the progressive impairment of SNAP in hexb^(−/−)mice (FIG. 13 b). In contrast, none of the different HDAd treatmentscould slow down the deterioration in compound muscle action potential(CMAP) in any of these animals (FIG. 14 b).

A functional neurological behavior test was performed by measuring thetime needed for the mice to remove an adhesive tape on their hindpaw(Fleming et al., 2004). In hexb^(−/−) mice, the time of first contactfor tape removal goes up progressively with age. Treatment with WF-hexbhad no effect on first contact time while DRG1-hexb treatmentsignificantly shortened this time and slowed down the deterioration inthis parameter (FIG. 13 c), at 4 and 8 weeks. The time required forsuccessful tape removal was also improved by DRG1-hexb, but not WF-hexb,at 4 weeks; however, the beneficial effect of the treatment on thisparameter, which depends also on relatively intact motor function,disappeared after 8 weeks (FIG. 13 d).

The structural integrity of DRG neurons was characterized histologically8 weeks after vector injection. By Niss1 staining, DRG neurons inhexb−/− mice treated with DRG1-hexb were similar to those in wild typemice, though the staining in hexb−/− mice was slightly less intense thanfor wild type mice (FIGS. 13 f,h). This is consistent with the resultsof enzyme histochemistry shown in FIG. 12. In contrast, DRG neurons inthe empty or WF-hexb group showed much lighter staining, suggestingneuronal degeneration (FIGS. 13 e,g). In periodic acid Schiff (PAS)staining, granular formation (FIGS. 13 i,m,k,o, arrows) was evident inmany DRG neurons in mice treated with the empty or WF-hexb vector, whilesuch accumulation of carbohydrate macromolecules was not detected in theDRG1-hexb-treated or hexb^(+/+) group (FIGS. 13 j,n,l,p).

Example 7 Significance of Certain Embodiments of the Invention

Sensory neuronopathies affecting DRG neurons that manifest as pain,hyperalgesia, hypo- and anesthesia or ataxia can be caused by genetic,nutritional, metabolic, inflammatory or neoplastic processes. A strategythat enables the efficient delivery of therapeutic transgenesspecifically to DRG neurons would represent a significant advance in thetreatment of DRG neuronal disorders, because therapeutic measures aimedat the underlying disease process only often fails to reverse theneuronopathy (Sghirlanzoni et al., 2005; Kuntzer et al., 2004).

In an exemplary embodiment of the invention, this problem was addressedby engineering fiber-modified HDAd that specifically targets DRG neuronsin vivo. HDAd was selected as the vector because of its large capacity,negligible toxicity, prolonged transgene expression in the absence ofintegration which minimizes its potential tumorigenic effects (Oka andChan, 2005). The target tissue specificity of an HDAd is determined bythe specificity of the helper virus (HV). By using a HV ablated forbinding to primary attachment receptors in the fiber (KO1S* mutation) asa platform, the inventors inserted into the HI loop DRG neuron-homingpeptides isolated by phage display and biopanning (Oi et al., 2008).Mutations were introduced in CAR and putative HSPG binding sites thatdetargeted Ad from 293 cells (Kritz et al., 2007 and DRG neurons. WhenAd was into the subarachnoid space, modified Ad was also detargeted fromDRG neurons in vivo. For subgroup C Ad, including Ad5, infection of Adin vitro occurs first by direct attachment of the fiber to the CAR,followed by internalization through αV integrin via the RGD motif ofpenton bases, or by interaction with HSPG (Dechecchi et al., 2001).However, IV Ad5 directly binds to coagulation factors via interactionbetween the FX Gla domain and hypervariable regions of the Ad5 hexonsurface (Waddington et al., 2008; Kalyuzhniy et al., 2008), and issubsequently internalized by binding to LRP and/or HSPG in the liver(Shayakhmetov et al., 2005; Parker et al., 2006). Koizumi et al. havereported that Ad ablated for binding to CAR, αV integrin and HSPG withinthe FG loop in the fiber knob, and Ads that carry a mutation of the ABloop and substitution of the fiber shaft derived from Ad type 35 havemarkedly reduced tropism to the liver; the modifications also attenuatedthe toxicity as defined by IL-6 secretory response after IV injection(2006). The penton and the RGD motif were left intact, as the RGD motifwas thought to be needed for endocytosis and endosomal escape (Wickhamet al., 1993; Shayakhmetov et al., 2005). Ablation for CAR and HSPG wassufficient to blunt natural tropism. In specific embodiments of theinvention, the direct subarachnoid delivery of the vector, a routineprocedure in clinical practice that also eliminates any directinteraction of Ad with blood components, is useful.

Chronic toxicity associated with early generation Ads has been largelyeliminated in HDAd; however, the acute inflammatory response caused byinnate immunity against capsid proteins was reported to be similar amongthe different types of Ad40. In certain embodiments of the presentinvention, markedly reduced IL-6 and TNF-α levels were identified afterintrathecal HDAd. It is intriguing that the IL-1β response was notgreatly attenuated by HDAd as compared with the HVs. An increase inIL-1β is an early response to both Ad and HDAd (McCaffrey et al., 2008)and is considered upstream to IL-6 and TNF-α. Intrathecal HDAd has beenreported to invoke neither systemic or local toxicity, nor aCNS-specific immune reaction (Butti et al., 2008). Therefore, the lowtoxicity found in specific embodiments of the invention are in part dueto presence of few immune cells in CSF, in certain aspects. In addition,use of detargeted HDAd-KO1S* and HDAd-DRG1 (which is also based onKO1S*) was associated with a 2-fold reduction of IL-6 at 3 and 24 hafter vector injection, an observation consistent with previous reportsshowing attenuated cytokine response by Ad detargeted for naturalreceptors (Koizumi et al., 2006).

Three exemplary peptide ligands were tested for their ability to targetDRG neurons. Although surface proteins interacting with these peptideshave not been identified, DRG1 and DRG3 appear to bind to small neuronswhile DRG2 binds to large neurons (Oi et al., 2008), in certain aspectsof the invention. HVs containing these peptide ligands increased theefficiency of transduction of DRG neurons in vitro as well as in vivo(FIG. 4). DRG1 was the most potent and transduction of DRG neurons byHV-DRG1 was more than 100-fold more efficient than HV-WF. This DRGtargeting HV had a profound effect on transgene expression. LacZexpression induced by HV-DRG1 at day 60 was still maintained at a levelsimilar to that by HV-WF at day 5. Despite an enhanced transductionefficiency of the targeting vector, there was no apparent effect oftargeting on decay of transgene expression (FIG. 10 a). This is notunexpected, however. Although tissue or cell targeting plays animportant role in efficient transduction, it does not influenceintracellular processing or degradation of vector chromosome. Phagedisplay is a powerful technique to identify homing peptides to specificin vivo molecular zip codes. DRG1 appears to support vectorinternalization and endosome escape, which does not always happen(Fechner et al., 1999). Phage is a small molecule compared to Ad and theaccess of bulky Ad particle could be hampered by physical and biologicalbarriers (Fechner et al., 1999). In fact, although IT HV-DRG1efficiently transduces DRG neurons, IV HV-DRG1 fails transduce DRGneurons in vivo.

Despite the many human diseases affecting DRG neurons, few mouse modelsof pure sensory neuron dysfunction are available. hexb−/− mice wereused, which exhibits global neurological impairment at 3 months,followed by ataxia, bradykinesia, impaired motor activity and balance.By 18 weeks, hind-limb movement is lost and they die before 20 weeks(Sango et al., 1995; Sango et al., 2002). As in vivo measurements ofsensory function have not been reported in this mouse model, theoccurrence of peripheral sensory impairment in this disorder was firstdocumented using sensory nerve conductions studies and mixedsensory/motor function (tape removal) tests (FIGS. 13 c,d). A singleinjection of DRG neuron targeting HDAd expressing Hexb by lumbarpuncture into the CSF increased Hex activity in DRG neurons and restoredperipheral sensory function but not motor neuron function in hexb−/−mice. In histological analysis, Hexb immunoreactive protein was readilydetected in DRG neurons of mice treated by HDAd-DRG1-hexb but not thoseby HDAd-WF-hexb. Very few PAS+ granular formation in the cell body(representing ganglioside accumulation) was seen in theHDAd-DRG1-hexb-treated mice. Niss1 stain was strongly positive inHDAd-DRG1-hexb group similar to that seen in hexb+/+ mice. It isimportant to note that HDAd-DRG1-hexb treatment conferred protection tosensory neuron function but had no detectable effect on motor nervefunction. When nerve function was assessed by the tape removal test,deterioration in first contact time was significantly improved by thetreatment at both 4 and 8 weeks, because sensory function was dominantin this behavior test. In contrast, the tape removal time, which isdetermined by a combination of sensory and motor functions, waspartially corrected only at 4 weeks but not at 8 weeks. Despite a markedimprovement in sensory nerve conduction, the behavior tests were stillquite abnormal because performance in these tests requires the presenceof intact peripheral and central nervous systems and the treatment hadno effect on the latter. Therefore, use of the hexb−/− mice enabled oneto show that DRG neuron targeted gene therapy specifically restores DRGfunction without affecting motor nerve, spinal cord or brain function.

There are many clinical applications of the present invention.Neuropathic pain occurs commonly among patients with various metabolicand inflammatory disorders. For example, diabetic neuropathy is the mostcommon chronic complication of diabetes, affecting millions of people inthe United States (Sadosky et al., 2008). Many of these individualssuffer neuropathic pain caused by sensory neuron dysfunction (Yasuda etal., 2003). Patients with ganglionopathies caused by autoimmune diseasesor drug toxicities also commonly experience severe neuropathic pain(Kuntzer et al., 2004). Treatment of these and other sensoryneuronopathies by intrathecal DRG-targeting HDAd that expressneurotrophic factors or analgesic peptides is a useful application ofthe invention. It has been reported that the immune response to foreignmaterial injected IT is attenuated, which may allow for the repeatadministration of HDAd via this route for repeated treatments (Muruve etal., 2004; Butti et al., 2008). HDAd can be administered repeatedly evenwhen it is given by the IV route, as long as one employs HVs ofdifferent serotypes (Kim et al., 2001; Morral et al., 1999). Finally,the non-integrating nature of HDAd is an advantage in that it minimizesthe chance of genotoxicity and induced tumorigenesis (Oka and Chan,2005).

In the present invention, there are methods and compositions for a newstrategy to produce HDAd that specifically target DRG neurons. Oneadvantage of this approach is that HDAd expressing therapeutic gene madeby a generic HV can be retargeted to tissues or cell types of interestby amplification with HV containing targeting ligands. HDAd andfiber-modified HV are separately produced and combined only at the finalamplification step. In certain aspects of the invention, one cancustomize HDAd delivery to treat specific diseases and organ systems.Clinical trials using early generations Ads have been ongoing for years(Tolcher et al., 2006; Chiocca et al., 2008).

Example 8 Exemplary Methods and Materials

Cell culture. To generate Ad5 wild type fiber expressing 293 cells(293-fiber), lentiviral vector mediated gene transfer was employed. Ad5fiber was subcloned by PCR from AdEasy vector using the followingprimers: 5′ upstream primer, 5′-GGATCCGCCACCATGAAGCGCGCAAGACCGTC-3′ (SEQID NO:88) and 3′ downstream primer, 5′-GCGGCCGCTTATTCTTGGGCAATGTATG-3′(SEQ ID NO:89). They contained artificial cloning sites (underlined) andthe Kozak sequence (bold faced). The Ad5 fiber cDNA was then subclonedinto the pHIV-EF-1-puro (a first generation SIN vector modified frompHIV-AP; Sutton et al., 1998). LV-Ad5-fiber was produced bycotransfection of LV shuttle vector and pME-VSVG into 293T cells bystandard calcium phosphate precipitation method (FIG. 5 a). 293-fibercells were established by selection on puromycin (1 μg/ml). 293,293-fiber and 293Cre66 cells were maintained in α-MEM supplemented with10% FBS and antibiotic-antimicotics (Invitrogen). Puromycin (1 μg/ml)and G418 (0.8 mg/ml) were included in the media for 293-fiber and293Cre66, respectively. DRG neurons were isolated from C57BL/6 mice oneday before experiments and cultured under F-12 with 10% FBS as previousdescribed (Oi et al., 2008). Infection was performed in non-serum mediaat 37° C. for 30 min-1 hour in a CO₂ incubator.

Construction of fiber-modified first generation helper virus (HV). Toconstruct fiber-modified HV using AdEasy system (Stratagene) (FIG. 3 a),a 2-kb fragment containing 1.7-kb NdeI/PmeI fragment coding for Ad5fiber was first subcloned into pCR2.1-TOPO (Invitrogen) by PCR usingpDV153-KO1S* as a template (pCR2.1-fiber-KO1S*). pDV153-KO1S* is aderivative of pDV137, but harboring the S* mutation (mutation of theputative HSPG-binding region) and the unique BspEI site in the HI loop33(FIG. 4 a). Three exemplary peptide ligands targeting DRG neuronsisolated by biopanning of phage display (Oi et al., 2008) were thensubcloned into the BspEI site of pCR2.1-fiber-KO1S*, resulting inpCR2.1-DRG1, -DRG2 and -DRG3. The 1.7 kb NdeI/PmeI fragment was excisedfrom these three vectors and pCR2.1-fiber-KO1S* and then subcloned intopDV153-KO1S* vector. The 7 kb SpeI/PacI fragment from these four cloneswere subcloned into pAdEasy-1 vector (Stratagene). To make fibermodified HV, the inventors modified pShuttle (Stratagene) to have thepackaging signal (φ) flanked by loxP sites with the MluI site downstreamof the second loxP site for cloning purposes (pShuttle-loxP-φ-loxP). Toinsert two reporter genes in HV, the inventors first subcloned both 5.2kb βgeo cassette and 2.2 kb BamHI/BglII fragment of RLCMV (Promega)containing Rluc expression cassette into pLPBL-1. The resulting dualgene expression cassette was excised by AscI digestion and subclonedinto the MluI site of pShuttle-loxP-φ-loxP(pShuttle-loxP-φ-loxP-βgeo-RLCMV). HV plasmids were generated byhomologous recombination in BJ5183 cells (Stratagene). HVs targeting DRGneurons (HV-DRG1, -DRG2 and -DRG3) and detargeted HV (HV-KO1S*) wereproduced by transfection into 293-fiber cells. HV-WF was produced on 293cells. After production of HVs, these were tested the excision of thepackaging signal by 293-Cre cells. Infection of 293-Cre with HVs removedthe packaging signal, resulting in 262 bp PCR product instead of 460 bpproduct from the intact HV (FIG. 15 a). The excision of the packagingsignal was also confirmed by DNA sequence analysis (FIG. 15 b).

Construction of helper-dependent adenoviral (HDAd) vectors. The cDNA ofcre recombinase was cloned by PCR using pBS185 vector (Invitrogen) astemplate and the following PCR primers: 5′ upstream primer,5′-TTAATTAAGCCACCATG TCCAATTTACTGACCGTACAC-3′ (SEQ ID NO:90) and 3′downstream primer, 5′-GCGGCCGCCTAATCGCCATCTTCCAGCAG-3′ (SEQ ID NO:91).They contained artificial cloning sites (underlined) and the Kozaksequence (bold faced). The hexb cDNA was cloned from C57BL/6 mouse braininto pCR2.1-TOPO (Invitrogen) by RT-PCR using the following primers: 5′upstream primer, 5′-ATGCCGCAGTCCCCGCGTA-3′ (SEQ ID NO:92) and 3′downstream primer, 5′-CTGGAATGCTGTAGACGTCTGTC-3′ (SEQ ID NO:93). ThesecDNAs were subcloned into pBOS vector that contains EF-1 promoter andrabbit β-globin polyadenylation signal on pLPBL-1 backbone. Theseexpression cassettes and βgeo-RLCMV described above were excised by AscIdigestion and then subcloned into pΔ28 vectors (Oka et al., 2001). AllHDAd vectors were first made by the published method using HV-WF and293Cre66 cells (FIG. 5 b) (Oka and Chan, 2005). Fiber-modified HDAdvectors were made by coinfection of HDAd vector and fiber-modified HV asoutlined in FIG. 3.

Mice. 8-12 week old male C57BL/6 male, B6; 129-Gt(ROSA)26Sor^(tm2Sho)/J(Rosa-GFP) and hexb+/− mice, a model of Sandhoff disease were purchasedfrom the Jackson Laboratory. Homozygous hexb−/− and wild-type mice(hexb+/+) were generated by mating hexb+/− mice and the litter mateswere used for studies. Genotyping was carried out according to theprotocol posted on the home page of The Jackson Laboratory. All animalswere housed, fed water and mouse chow ad libitum, and maintained under a12-h light and dark cycle. All experimental protocols were performedaccording to the guidelines of Institutional Animal Care and UsageCommittee at Baylor College of Medicine.

Luciferase assay in vitro and in vivo. For in vitro studies, theinventors plated 293 cells and isolated DRG neurons from C57BL/6 miceone day before infection and infected with HVs at 1,000 viral particles(vp)/cell. Next day, cells were harvested in a lysis buffer suppliedwith the luciferase assay kit (Promega) and the lysates were assayed forluciferase activity as described in manufacture's protocol. For in vivostudies, 1×10⁸ vp of each vector were injected into mice throughsubarachnoid space at lumbar level. Mice were euthanized 5 days aftervector treatment and brain, spinal cord, DRG and sciatic nerve wereisolated. Tissues were homogenized with lysis buffer and thesupernatants were assayed for luciferase activity.

X-gal staining of cells and nervous tissues. For in vitro studies, cellswere fixed with fixation solution supplied in beta-gal staining kit(Invitrogen) one day after infection and incubated with X-gal as permanufacture's protocol. For in vivo studies, mice were transcardiacallyfixed with 4% paraformaldehyde one day after vector injection andisolated nervous tissues were incubated with X-gal for 4 hours. Alltissues were observed under standard microscope and stereomicroscope.Several pictures for brain and spinal cord were combined to show thewhole central nervous system. Subsequently, 10 μm thick cryosectionswere prepared.

Quantitative RT-PCR. Total cellular RNA was extracted by Trizol(Invitrogen) and treated with DNase I digestion. After reversetranscription using oligo dT, mRNA was quantified by real-time PCR usingiQ SYBR Green Supermix (Bio-Rad). The following exemplary primers wereused: Lac-Z; 5′ upstream primer, 5′-GTTGCAGTGCACGGCAGATACACTTGCTGA-3′(SEQ ID NO:94) and 3′ downstream primer,5′-GCCACTGGTGGGCCATAATTCAATTCGC-3′ (SEQ ID NO:95), cre-recombinase; 5′upstream primer, 5′-GCATTACCGGTCGATGCAACGAGTGATGAG-3′ (SEQ ID NO:96) and3′ downstream primer, 5′ GAGTGAACGAACCTGGTCGAAATCAGTGCG-3′ (SEQ IDNO:97), GFP; 5′ upstream primer, 5′-GCACGACTTCTTCAAGTCCGCCATGC-3′ (SEQID NO:98) and 3′ downstream primer, 5′-GCGGATCTTGAAGTTCACCTTGATGCC-3′(SEQ ID NO:99), hexb; 5′ upstream primer, 5′-ACAGTTGCAGAAGCTCCTGGT-3′(SEQ ID NO:100) and 3′ downstream primer, 5′-CCTCTATGAGGGAATCTTGGAG-3′(SEQ ID NO:101), β-actin; 5′ upstream primer, 5′-GACGGCCAGGTCATCACTAT-3′(SEQ ID NO:102) and 3′ downstream primer, 5′-CTTCTGCATCCTGTCAGCAA-3′(SEQ ID NO:103). The detection was carried out with Mx3000P QPCR system(Stratagene) and the results were analyzed by MxProQPCR software(Stratagene). All data were normalized to β-actin.

Immunoblots. DRG and 293-fiber cells were lysed with RIPA buffer [150 mMNaCl, 2 mM EDTA, 1% nonidet P-40, 1% sodium deoxycholate, 0.1% sodiumdodecyl sulfate (SDS), 50 mM NaF, 10 μg/mL aprotinin, 10 μg/mLleupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM Tris-HCl buffer, pH7.4] and were centrifuged at 12,000×g for 20 min at 4° C. to collect thesupernatant. 20 μg of proteins were separated by 7.5-15% SDS-PAGE andwere transferred to a polyvinylidene difluoride filter (ImmobilonMillipore, Bedford, Mass.). The blots were incubated overnight at 4° C.with rabbit anti-Cre antibody (1:1000, Novagen), mouse monoclonalanti-GFP antibody (1:1000, Clontech) or anti-adenovirus fiber (1:1000,ab3233, Abcam). Immunoreactive proteins were detected by enhancedchemiluminescence (PIERCE Biotechnology) after incubation with eitherhorseradish peroxidase-conjugated anti-rabbit immunoglobulin oranti-mouse immunoglobulin (1:2000, Bio-Rad).

ELISA for cytokines in CSF. Cerebrospinal fluid (CSF) was collected fromcistern magna at 3 h, 24 h, 3 days, 7 days and 14 days after vectorinjection. IL-β, IL-6 and TNF-alpha levels were determined using anELISA kit (eBioscience).

Hexosaminidase enzyme assay. Total β-Hexosaminidase activity wasmeasured using4-methylumbelliferyl-2-acetamido-2-deoxy-β-D-glucopyranoside (4-MUG;Sigma, St. Louis, Mo.) as the substrate. β-Hexosaminidase A activity wasassayed using4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-β-D-glucopyranoside(4-MUGS; Calbiochem, San Diego, Calif.). β-Hexosaminidase B activity wasdetermined by subtracting β-Hexosaminidase A activity from totalHexosaminidase activity (Yamaguchi et al., 2003). Enzyme activity isexpressed as nmol/mg protein/hr.

Histological analysis of GFP expression in ROSA-GFP mice. For GFPexpression analysis, nervous tissues were directly observed underfluorescent microscope after dissections. The 10 μm cryo sections wereprepared from DRG and mounted with VECTASHIELD mounting medium with DAPI(Vector Laboratories).

Histological analysis of hexb−/− mice. Mice were perfused transcardiallywith cold saline followed by 4% paraformaldehyde, 0.05% glutaraldehyde,0.5% picric acid and 0.01 M phosphate buffer (pH 7.4) solution. L1-5 DRGtissues were removed and 10-μm thick cryosections were incubated withgoat anti-hexb antibody (1:100, Santa Cruz Biotechnology) overnight at4° C. Next day, the sections were incubated with rhodamine labeleddonkey anti-goat IgG and mounted with VECTASHIELD mounting medium withDAPI (Vector Laboratories). Other sections from vector treated mice werealso used for periodic acid schiff (PAS) (PAS staining system, Sigma)and Niss1 stain with cresyl violet (Sigma). For enzymatic histochemicalanalysis of hexosaminidase, DRG were immersed in Baker fixative solution(4% paraformaldehyde, 1% anhydrous calcium chloride, pH7.2) for 1 h at4° C., and then immersed into gum-sucrose solution (14% antisepticthymol 100, 0.14% gum arabic, 1.25 M saccharose) for 24 h at 4° C. 10 μmcryosections were incubated in working solution containing 0.5 mMnaphthol-AS-BI-D-N-acetyl-β-glucosaminide (Sigma) and 3.6 mM Fast GarnetGBC sulfate salt (Sigma) at pH 5.2 for 30 min at 37° C. and thencounterstained with 1% methyl green for nuclei.

Electrophysiological studies and behavior tests. Nerve conductionstudies (NCS) were performed with CADWELL SIERRA 6200A (CadwellLaboratories) under anesthetization at 30° C.-32° C. temperature. Motornerve conduction study was performed in the sciatic nerves as describedpreviously (Terashima et al., 2005). For sensory nerve conductionstudies, sural nerves were stimulated in the distal site at ankle jointlevel and were recorded in proximal site (Terashima et al., 2005).Adhesive removal tests were performed as neurological behavior tests.Small adhesive stimuli (Avery adhesive labels, one-quarter inch round)were placed on the hindpaw of the mouse, and the time to make contactand remove the sticker was recorded. To remove the stickers, animalswould use both forelimbs toward their feet and swipe off the stimuluswith both forepaws. Each mouse received three trials and had aninter-trial interval of at least 3 min. All testing was performed in theanimal's home cage (Fleming et al., 2004).

Statistical analysis. All data are expressed as means±S.D. All in vitroexperiments were performed in triplicate in at least three independentexperiments. In vivo analyses were performed using five mice/groupunless specified. The Student's unpaired two tailed t-test was used tocalculate statistical significance. Data were considered significant atp<0.05.

REFERENCES

All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the invention asdefined by the appended claims. Moreover, the scope of the presentapplication is not intended to be limited to the particular embodimentsof the process, machine, manufacture, composition of matter, means,methods and steps described in the specification. As one will readilyappreciate from the disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized. Accordingly, the appended claims areintended to include within their scope such processes, machines,manufacture, compositions of matter, means, methods, or steps.

What is claimed is:
 1. An isolated peptide of SEQ ID NO:3.
 2. Thepeptide of claim 1, comprised in a pharmaceutically acceptableexcipient.
 3. The peptide of claim 1, linked to a protein or a liposome.4. The peptide of claim 3, wherein the protein is a therapeutic protein.5. The peptide of claim 4, wherein the therapeutic protein is NGF, EGF,FGF, BDNF, IGF1, CTNF, PDNF, VAD, DEVD, NGN1, NGN2, NGN3, RUNX3, or asignal peptide.
 6. The peptide of claim 3, wherein the protein ispresent on the surface of a helper-dependent adenoviral particle.
 7. Thepeptide of claim 6, wherein the particle is defined as furthercomprising a therapeutic polynucleotide.
 8. The peptide of claim 7,wherein the therapeutic polynucleotide is selected from the groupconsisting of NGF, EGF, FGF, BDNF, IGF1, CTNF, PDNF, VAD, DEVD, NGN1,NGN2, NGN3, RUNX3, IL-10, anti-TNFα, EPO, or prepro-β endorphin.
 9. Apolynucleotide encoding the peptide of SEQ ID NO:3.